BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS Pages 937-944
Vol. 183, No. 3, 1992 March 31, 1992
GENE
EXPRESSION
AND IMMUNOHISTOCHEMICAL
LOCALIZATION OF BASIC FIBROBLAST GROWTH FACTOR IN RENAL CELL CARCINOMA
Jiro Eguchi*, Koichiro Nomata, Shigeru Kanda, Tsukasa Igawa, Masakatsu Taide, Shigehiko Koga, Fukuzo Matsuya, Hiroshi Kanetake and Yutaka Saito Department of Umlogy, Nagasaki Univemity School of Medicine, Nagasaki 852, Japan Received February 8, 1992
SUMMARY: Renal cell carcinoma is known as a neoplastic condition of renal tubular cells and usually shows a hypervascular tumor in angiographic examination. We examined the presence of basic fibroblast growth factor (bFGF) in human renal cell carcinoma. To determine if alterations in bFGF gene expression are present in human renal cell carcinoma, paired samples of normal and neoplastic renal tissue from 6 patients were analyzed for bFGF mRNA content by Northern blot hybridization. In 4 out of 6 patients, tumor tissue expressed bFGF mRNA 2 to 4 times greater than corresponding normal tissue. Two patients showed minimal elevation of tumor bFGF mRNA. The localization of bFGF in the renal cell carcinoma tissue was also examined using immunohistochemical staining, and it was found that bFGF was positively stained at the nuclei of tumor cells and the cell surface. These results suggest that increased expression of bFGF may be associated with neoplastic growth in renal tubular epithelial cells and neovascularization. © 1992 A c a d e m i c
Press,
Inc.
Most renal cell carcinomas show increased vascularity.
The most
characteristic angiographic finding is the presence of "tumor" vessels (1). Therefore, we thought that some growth factors play an important role in its neovascularization. Fibroblast growth factor (FGF), which exists under two closely related formsl one basic and the other acidic, has been shown to trigger the proliferation and differentiation
of a wide variety of mesoderm and
whom correspondence should be addressed. Abbreviations: EGF, epidermal growth factor; FGF, fibroblast growth factor; PD-ECGF, platelet-derived endothelial cell growth factor; TAF, tumor angiogenesis factor; TGF, transforming growth factor. * TO
937
0006-291X/92 $1.50 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
neuroectoderm-derived cells in particular that of endothelial cells derived either from large vessels or from capillaries (2). Recently, it has been reported that FGF plays an important role in the regulation of some epithelial cell growth (3-5). Basic FGF (bFGF) is a single-chain polypeptide composed of 146 amino acids with a molecular weight in the range of 15-16 kDa (2) and has 53% sequence homology to acidic FGF (aFGF) (6).
The nucleotide
sequences of rat, human and bovine bFGF show very high levels of conservation, especially in their coding regions (88.7% identity between rat and human, 88.5% between rat and bovine and 94.9% between bovine and human) (7). The potential importance of FGF in tumor biology is supported by the discovery of two oncogenes, hst-1 and int-2, in human tumors encoding proteins which are structurally related to the FGF family of growth factors (8). It has been reported that bFGF mRNA expression in gastric carcinoma is higher than in corresponding normal gastric mucosa (9). Murphy et al. reported that bFGF transcript levels are elevated more in schwannoma samples than the average level of expression in benign meningioma samples (10).
On the
other hand, the observation that bFGF is mitogenic for cultured human renal carcinoma cells (11) supports the possibility that inappropriate expression of bFGF may contribute to the growth of renal cell carcinoma. In this study, we found that bFGF gene expression in some renal cell carcinoma tissues is greater than in corresponding normal renal tissues, and examined the distribution of bFGF in renal cell carcinoma tissue. MATERIALS AND METHODS
Tissues: Tissue samples were obtained at the time of surgery from 6 patients with previously untreated renal cell carcinoma. Tissues were taken from the tumor and from adjacent normal kidney tissue at the time of surgery, after radical nephrectomy, and were frozen quickly on dry ice and stored at -80oC until analysis. The histology of each specimen was evaluated upon hematoxylin-eosin staining of tissue adjacent to that used for RNA extraction. RNA extraction and Northern analysis: The methods for extraction of RNA and Northern analysis were described previously (12). Briefly, the frozen tissues were homogenized in guanidine isothiocyanate buffer (4 M guanidine isothiocyanate, 25 mM sodium citrate pH 7.0, 0.5% sarkosyl, 0.1 M beta-mercaptoethanol) and the homogenate was placed on a CsCI cushion (5.7 M CsCI, 0.1 M ethylenediamine tetraacetic acid, disodium salt (EDTA) pH 7.0). Total RNA was pelleted by centrifugation for 18 h at 44,400 g. The RNA was solubilized in distilled water. 938
Vol. 183, No. 3. 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
Total RNA (20 ~g per lane) was separated on 1.0% agarose-2.2 M formaldehyde gels and transferred to Hybond-N membranes (Amersham, Tokyo, Japan), followed by baking at 80oC for 2 h and incubation in prehybridization buffer (50% deionized formamide, 4 X SSPE (1 X SSPE: 10 mM sodium phosphate pH 7.0, 10 mM EDTA, 180 mM NaCI), 1% sodium - dodecyl sulfate (SDS), 0.5% skim milk, 0.1 mg/ml yeast RNA, 0.1 mg/ml denatured salmon sperm DNA) at 42oC for 16 h. The bFGF-cDNA probe (7) was kindly provided by Takeda Chemical Industries, Osaka, Japan. The probe was labelled using a random prime labelling system (Amersham, Tokyo, Japan) with [alpha 32p]-dCTP (3,000 Ci/mM: Amersham, Tokyo, Japan) and denatured. The membranes were then hybridized with probes [1-2 X 107 cpm/ml] in hybridization buffer (50% deionized formamide, 4 X SSPE, 10% dextransulfate, 1% SDS, 0.5% skim milk, 0.2 mg/ml yeast RNA) at 42oC for 18 h, and were washed four times in 2 X SSC (1 X SSC: 150 mM NaCI, 15 mM trisodium citrate pH 7.0) and 0.1% SDS at room temperature for 5 min and twice in 0.1 X SSC and 0.1% SDS at 50oC for 15 min, followed by exposure on Konica X-ray film (Konica, Tokyo, Japan) with two intensifying screens at -80oC for 4 days. Autoradiographs were scanned for quantification with an F-808 Cosmo densitometer (Cosmo Co., Ltd., Tokyo, Japan). Immunohistochemistry: Sections cut at 3-5 ~m thickness from a paraffin block of formalin fixed tissue were deparaffinized for use. First, the tissues were incubated with 0.3% H202 in absolute methanol for 30 min and then washed with buffer A (phosphate buffered saline, pH 7.2, containing 0A% Tween 20 and 0.1% bovine serum albumin), whereafter it was again incubated with 5% bovine serum albumin for 20 min at room temperature. Next, the bovine serum albumin was removed and the tissues were incubated with anti-bFGF rabbit IgG (200 ~g/ml: R&D systems, Minneapolis, MN) or nonimmunized rabbit IgG (200 I~g/ml) for 120 min at 37oC. Following this, the sections were washed with buffer A and incubated with biotinylated antirabbit IgG (60 gg/ml) for 60 min at 37oC, then washed with buffer A and incubated in a 50-fold diluted streptoavidin-peroxidase complex for 30 min at room temperature. Finally, the sections were washed with buffer A and the peroxidase reaction was performed in a diaminobenzidine solution containing 0.025% COCI2 and 0.02% NiSO4. RESULTS bFGF mRNA expression in normal human kidney and renal cell carcinoma tissues:
The characteristics of patients with renal cell
carcinoma analyzed in this study are summarized in Table 1. The tumor stage was all I; and the grade of the tumor cells was I in five patients and II in one patient. All tumors showed hypervascularity, judged by renal angiography. The expression of bFGF mRNA in neoplastic and normal kidney tissue obtained from the 6 patients with renal cell carcinoma is shown in Fig. 1. A single bFGF transcript of approximately 7.0 kilobases (kb), similar in size to bFGF mRNA from other sources (10,13), was detected in normal and tumor samples from 6 patients. Low constitutive levels were detected in all normal 939
V o l . 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1. Clinical Features and mRNA Levels of bFGF in 6 Renal Cell Carcinomas
Case No.
Relative expression a) T/N
Sex
Age
Grade
Stage
1
M
59
I > II
!
2.26
2
M
57
I
I
3.89
3
F
68
!
I
3.86
4
F
63
I
I
2.88
5
F
51
II
I
1.26
6
M
63
I
I
1.41
a) Ratio of direct densitometric measurement of autographic signals from Northern blot hybridization in renal cell carcinoma tissues (T) and corresponding normal tissues (N). kidney tissues, and renal cell carcinoma tissues expressed higher levels of m R N A in the majority of samples.
As measured by densitometory, 4 of 6
samples demonstrated a 2- to 4-fold increase in bFGF mRNA and the other 2 samples showed a minimal elevation of bFGF mRNA (Table 1).
Etidium
bromide staining revealed that nearly equal amounts of RNA were present in each lane (Fig. 1).
Immunohistochemical carcinoma
tissue:
staining
of
Immunohistochemical
bFGF
in
renal
cell
staining of bFGF was also
performed in order to clarify the localization of bFGF in renal cell carcinoma, 1
Case N
2 T
N
3 T
N
4 T
N
b FGF
5 T
N
6 T
N
T
7.0 Kb
28 S
Et Br staining
18 S
Fig, 1. bFGF mRNA expression in normal human kidney and renal cell carcinoma tissues, bFGF mRNA levels determined by Northern
analysis in renal cell carcinoma surgically removed tumor specimens (T) and corresponding normal tissues (N). Numbers above the lanes are sample numbers. The intensities of autoradiographic signals were determined by densitometry. Etidium bromide staining revealed that nearly equal amounts of RNA were present in each lane. 940
Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. 2. Immunohistochemical staining of bFGF in renal cell carcinoma tissue. Details of the experimental conditions were as
described in "Materials and Methods". The immunoreactivity is confined to the nucleus of tumor cells (arrow head). A: nonimmunized rabbit IgG; B: antibFGF rabbit IgG. Scale bar=10 I~m.
which is shown in Fig. 2, The results showed that there was a strong bFGF immunoreactivity at the nuclei of the tumor cells, and the cell surface was also positively stained. In normal renal tissue, little immunoreactivity was present at the glomerular cells and the tubular cells (data not shown). These findings suggest that increases of bFGF mRNA cause the expression of bFGF in renal cell carcinoma tissue, and that bFGF may regulate the tumor cell growth (nuclear FGF) or the neovascularization (cell surface FGF). 941
Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS DISCUSSION
The conversion of a normal cell to a malignant one is a multifactorial process (14). One of the important processes is deregulation of the growth factor and growth factor receptor gene expression.
For example, recent
studies of renal cell carcinoma suggest that overexpression of EGF receptor, TGF-c~ and TGF-131 genes may play an important role in either initiation or progression of malignant transformation (15,16). On the other hand, bFGF is known as a potent angiogenic factor and has been purified from various tissues, including kidney (17).
Shahabuddin et al. reported that tumor
angiogenesis factor (TAF) is present in renal cell carcinoma tissue (18). There are some angiogenic factors that have been already reported. Angiogenin has been isolated from the conditioned medium of a human colonic adenocarcinoma line (19), and Usuki et al. recently found that platelet-derived endothelial cell growth factor (PD-ECGF) is produced by a human squamous cell carcinoma cell line and two human anaplastic thyroid carcinoma cell lines (20).
However, angiogenin and PD-ECGF have not yet been identified in
renal Cell carcinoma tissue. In this study, we examined the expression of bFGF that might be related to the development of renal cell carcinoma. The bFGF mRNA transcripts of 7.0 kb were detected in all of the renal cell carcinoma examined (Fig. 1).
In
addition, the mRNA levels of bFGF in renal cell carcinoma were higher than corresponding normal renal tissue. To contribute to the elucidation of the role of bFGF
in the evolution
of renal cell carcinoma,
we performed
immunohistochemical staining of tumor tissue using anti-bFGF antibody, and found that immunoreactive bFGF was present in the nucleus and on the cell surface of the tumor cells (Fig. 2). This indicates that bFGF appears to relate to tumorigenesis of renal cell carcinoma.
Dell'Era et al. reported that
endogenous bFGF undergoes an intracellular sorting to the nucleus of the endothelial cell (21). We also present the immunohistochemical evidence on the presence of bFGF in the nucleus of renal cell carcinoma cells.
It may
suggest that nuclear FGF can promote the autocrine growth of tumor cells and 942
Vol. 183, No. 3, 1 9 9 2
BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS
cell surface FGF is able to stimulate the endothelial cell growth in a paracrine fashion.
However, there is no obvious explanation about a specific
mechanism of nuclear and cell surface FGF. Tanimoto et al. reported that bFGF mRNA expression in gastric carcinoma is higher than in corresponding normal gastric mucosa, especially, in scirrhus type (9).
All of the tumors used in our study showed
hypervascularity in the angiographic study and were low grade and low stage tumors, and no remarkable correlation between tumor character and clinical course was found. From our present data, it is suggested that bFGF may play an important role in progression of renal cell carcinoma, and bFGF may be the only TAF in renal cell carcinoma.
Further study of the signal transduction
pathway of bFGF in renal cell carcinoma is necessary in order to clarify the basic aspects of the biological character of human renal cell carcinoma.
ACKNOWLEDGMENTS We thank our branch hospital doctors for providing tissue samples and T. Shimogama, E. Taguchi, M. Yoshimoto, S. Honda and E. Suda for their technical assistance. This work was partially supported by the Grant-in-Aid for Scientific Research, the Ministry of Education, Science, and Culture, Japan.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
Levine, E., (1990) In Clinical Urography (H. M. Pollack, Ed. ), Vol. II, pp. 1256-1261, Saunders, Philadelphia. Gospodarowicz, D., Neufeld, G. and Schweigerer, L., (1986) Mol. Cell. Endocrinol., 46, 187-204. Crabb, J. W., Armes, L. G., Johnson, C. M. and McKeehan, W. L., (1986) Biochem. Biophys. Res. Commun., 136, 1155-1161. Kanda, S., Nomata, K., Saha, P. K., Nishimura, N., Yamada, J., Kanetake, H. and Saito, Y., (1989) Cell Biol. Int. Rep., 13, 687-699. Takahashi, K., Suzuki, K., Kawahara, S. and Ono, T., (1989) Int. J. Cancer, 43, 870-874. Esch, F., Ueno, N., Baird, A., Hill, F., Denoroy, L., Ling,N., Gospodarowicz, D. and Guillemin, R., (1985) Biochem. Biophys. Res. Commun., 133, 554-562. Kurokawa, T., Seno, M. and Igarashi, K., (1988) Nucleic Acids Res., 16, 5201. Yoshida, T., Miyagawa, K., Odagiri, H., Sakamoto, H., Little, P. F. R., Terada, M. and Sugimura, T., (1987) Proc. Natl. Acad. Sci. USA, 84, 7305-7309. Tanimoto, H., Yoshida, K., Yokozaki, H., Yasui, W., Nakayama, H., Ito, H., Ohama, K. and Tahara, E., (1991) Virchows Archiv B Cell Pathol., (in press). 943
Vol. 183, No. 3, 1 9 9 2
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
10. Murphy, P. R., Myal, Y., Sato, Y., Sato, R., West, M. and Friesen, H. G., (1989) Mol. Endo., 3, 225-231. 11. Nishimura, N., (1991)Act. Med. Nagasaki, (in press). 12. Nomata, K., Igarashi, H., Kanetake, H., Miyamoto, T. and Saito, Y., (1990) Urol. Res., 18, 251-254. 13. Mori, H., Maki, M., Oishi, K., Jaye, M., Igarashi, K., Yoshida, O. and Hatanaka, M., (1990) Prostate, 16, 71-80. 14. Heldin, C. H. and Westermark, B., (1984) Cell, 37, 9-20. 15. Sargent, E. R., Gomella, L. G., Belldegrun, A., Linehan, W. M. and Kasid, A., (1989) J. Urol., 142, 1364-1368. 16. Gomella, L. G., Sargent, E.R., Wade, T. P., Anglard, P., Linehan, W. M. and Kasid, A., (1989) Cancer Res., 49, 6972-6975. 17. Baird, A., Esch, F., Bohlen, P., Ling, N. and Gospodarowicz, D., (1985) Regul. Peptides, 12, 201-213. 18. Shahabuddin, S., Arnold, F., Costello, C, B. and Kumar, S., (1984) Br. J. Urol., 56, 490-494. 19. Fett, J. W., Strydom, D. J., Lobb, R. R., Alderman, E. M., Bethune, J. L., Riordan, J. F. and Vallee, B. L., (1985) Biochemistry, 24, 5480-5486. 20. Usuki, K., Heldin, N. E., Miyazono, K., Ishikawa, F., Takaku, F., Westermark, B. and Heldin, C. H., (1989) Proc. Natl. Acad. Sci. USA, 86, 7427-7431. 21. Dell'Era, P., Presta, M. and Ragnotti, G., (1991) Exp. Cell Res., 192, 505-510.
944
Vol. 183, No. 3, 1992 March 31, 1992
GENE
EXPRESSION
AND IMMUNOHISTOCHEMICAL
LOCALIZATION OF BASIC FIBROBLAST GROWTH FACTOR IN RENAL CELL CARCINOMA
Jiro Eguchi*, Koichiro Nomata, Shigeru Kanda, Tsukasa Igawa, Masakatsu Taide, Shigehiko Koga, Fukuzo Matsuya, Hiroshi Kanetake and Yutaka Saito Department of Umlogy, Nagasaki Univemity School of Medicine, Nagasaki 852, Japan Received February 8, 1992
SUMMARY: Renal cell carcinoma is known as a neoplastic condition of renal tubular cells and usually shows a hypervascular tumor in angiographic examination. We examined the presence of basic fibroblast growth factor (bFGF) in human renal cell carcinoma. To determine if alterations in bFGF gene expression are present in human renal cell carcinoma, paired samples of normal and neoplastic renal tissue from 6 patients were analyzed for bFGF mRNA content by Northern blot hybridization. In 4 out of 6 patients, tumor tissue expressed bFGF mRNA 2 to 4 times greater than corresponding normal tissue. Two patients showed minimal elevation of tumor bFGF mRNA. The localization of bFGF in the renal cell carcinoma tissue was also examined using immunohistochemical staining, and it was found that bFGF was positively stained at the nuclei of tumor cells and the cell surface. These results suggest that increased expression of bFGF may be associated with neoplastic growth in renal tubular epithelial cells and neovascularization. © 1992 A c a d e m i c
Press,
Inc.
Most renal cell carcinomas show increased vascularity.
The most
characteristic angiographic finding is the presence of "tumor" vessels (1). Therefore, we thought that some growth factors play an important role in its neovascularization. Fibroblast growth factor (FGF), which exists under two closely related formsl one basic and the other acidic, has been shown to trigger the proliferation and differentiation
of a wide variety of mesoderm and
whom correspondence should be addressed. Abbreviations: EGF, epidermal growth factor; FGF, fibroblast growth factor; PD-ECGF, platelet-derived endothelial cell growth factor; TAF, tumor angiogenesis factor; TGF, transforming growth factor. * TO
937
0006-291X/92 $1.50 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
neuroectoderm-derived cells in particular that of endothelial cells derived either from large vessels or from capillaries (2). Recently, it has been reported that FGF plays an important role in the regulation of some epithelial cell growth (3-5). Basic FGF (bFGF) is a single-chain polypeptide composed of 146 amino acids with a molecular weight in the range of 15-16 kDa (2) and has 53% sequence homology to acidic FGF (aFGF) (6).
The nucleotide
sequences of rat, human and bovine bFGF show very high levels of conservation, especially in their coding regions (88.7% identity between rat and human, 88.5% between rat and bovine and 94.9% between bovine and human) (7). The potential importance of FGF in tumor biology is supported by the discovery of two oncogenes, hst-1 and int-2, in human tumors encoding proteins which are structurally related to the FGF family of growth factors (8). It has been reported that bFGF mRNA expression in gastric carcinoma is higher than in corresponding normal gastric mucosa (9). Murphy et al. reported that bFGF transcript levels are elevated more in schwannoma samples than the average level of expression in benign meningioma samples (10).
On the
other hand, the observation that bFGF is mitogenic for cultured human renal carcinoma cells (11) supports the possibility that inappropriate expression of bFGF may contribute to the growth of renal cell carcinoma. In this study, we found that bFGF gene expression in some renal cell carcinoma tissues is greater than in corresponding normal renal tissues, and examined the distribution of bFGF in renal cell carcinoma tissue. MATERIALS AND METHODS
Tissues: Tissue samples were obtained at the time of surgery from 6 patients with previously untreated renal cell carcinoma. Tissues were taken from the tumor and from adjacent normal kidney tissue at the time of surgery, after radical nephrectomy, and were frozen quickly on dry ice and stored at -80oC until analysis. The histology of each specimen was evaluated upon hematoxylin-eosin staining of tissue adjacent to that used for RNA extraction. RNA extraction and Northern analysis: The methods for extraction of RNA and Northern analysis were described previously (12). Briefly, the frozen tissues were homogenized in guanidine isothiocyanate buffer (4 M guanidine isothiocyanate, 25 mM sodium citrate pH 7.0, 0.5% sarkosyl, 0.1 M beta-mercaptoethanol) and the homogenate was placed on a CsCI cushion (5.7 M CsCI, 0.1 M ethylenediamine tetraacetic acid, disodium salt (EDTA) pH 7.0). Total RNA was pelleted by centrifugation for 18 h at 44,400 g. The RNA was solubilized in distilled water. 938
Vol. 183, No. 3. 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
Total RNA (20 ~g per lane) was separated on 1.0% agarose-2.2 M formaldehyde gels and transferred to Hybond-N membranes (Amersham, Tokyo, Japan), followed by baking at 80oC for 2 h and incubation in prehybridization buffer (50% deionized formamide, 4 X SSPE (1 X SSPE: 10 mM sodium phosphate pH 7.0, 10 mM EDTA, 180 mM NaCI), 1% sodium - dodecyl sulfate (SDS), 0.5% skim milk, 0.1 mg/ml yeast RNA, 0.1 mg/ml denatured salmon sperm DNA) at 42oC for 16 h. The bFGF-cDNA probe (7) was kindly provided by Takeda Chemical Industries, Osaka, Japan. The probe was labelled using a random prime labelling system (Amersham, Tokyo, Japan) with [alpha 32p]-dCTP (3,000 Ci/mM: Amersham, Tokyo, Japan) and denatured. The membranes were then hybridized with probes [1-2 X 107 cpm/ml] in hybridization buffer (50% deionized formamide, 4 X SSPE, 10% dextransulfate, 1% SDS, 0.5% skim milk, 0.2 mg/ml yeast RNA) at 42oC for 18 h, and were washed four times in 2 X SSC (1 X SSC: 150 mM NaCI, 15 mM trisodium citrate pH 7.0) and 0.1% SDS at room temperature for 5 min and twice in 0.1 X SSC and 0.1% SDS at 50oC for 15 min, followed by exposure on Konica X-ray film (Konica, Tokyo, Japan) with two intensifying screens at -80oC for 4 days. Autoradiographs were scanned for quantification with an F-808 Cosmo densitometer (Cosmo Co., Ltd., Tokyo, Japan). Immunohistochemistry: Sections cut at 3-5 ~m thickness from a paraffin block of formalin fixed tissue were deparaffinized for use. First, the tissues were incubated with 0.3% H202 in absolute methanol for 30 min and then washed with buffer A (phosphate buffered saline, pH 7.2, containing 0A% Tween 20 and 0.1% bovine serum albumin), whereafter it was again incubated with 5% bovine serum albumin for 20 min at room temperature. Next, the bovine serum albumin was removed and the tissues were incubated with anti-bFGF rabbit IgG (200 ~g/ml: R&D systems, Minneapolis, MN) or nonimmunized rabbit IgG (200 I~g/ml) for 120 min at 37oC. Following this, the sections were washed with buffer A and incubated with biotinylated antirabbit IgG (60 gg/ml) for 60 min at 37oC, then washed with buffer A and incubated in a 50-fold diluted streptoavidin-peroxidase complex for 30 min at room temperature. Finally, the sections were washed with buffer A and the peroxidase reaction was performed in a diaminobenzidine solution containing 0.025% COCI2 and 0.02% NiSO4. RESULTS bFGF mRNA expression in normal human kidney and renal cell carcinoma tissues:
The characteristics of patients with renal cell
carcinoma analyzed in this study are summarized in Table 1. The tumor stage was all I; and the grade of the tumor cells was I in five patients and II in one patient. All tumors showed hypervascularity, judged by renal angiography. The expression of bFGF mRNA in neoplastic and normal kidney tissue obtained from the 6 patients with renal cell carcinoma is shown in Fig. 1. A single bFGF transcript of approximately 7.0 kilobases (kb), similar in size to bFGF mRNA from other sources (10,13), was detected in normal and tumor samples from 6 patients. Low constitutive levels were detected in all normal 939
V o l . 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1. Clinical Features and mRNA Levels of bFGF in 6 Renal Cell Carcinomas
Case No.
Relative expression a) T/N
Sex
Age
Grade
Stage
1
M
59
I > II
!
2.26
2
M
57
I
I
3.89
3
F
68
!
I
3.86
4
F
63
I
I
2.88
5
F
51
II
I
1.26
6
M
63
I
I
1.41
a) Ratio of direct densitometric measurement of autographic signals from Northern blot hybridization in renal cell carcinoma tissues (T) and corresponding normal tissues (N). kidney tissues, and renal cell carcinoma tissues expressed higher levels of m R N A in the majority of samples.
As measured by densitometory, 4 of 6
samples demonstrated a 2- to 4-fold increase in bFGF mRNA and the other 2 samples showed a minimal elevation of bFGF mRNA (Table 1).
Etidium
bromide staining revealed that nearly equal amounts of RNA were present in each lane (Fig. 1).
Immunohistochemical carcinoma
tissue:
staining
of
Immunohistochemical
bFGF
in
renal
cell
staining of bFGF was also
performed in order to clarify the localization of bFGF in renal cell carcinoma, 1
Case N
2 T
N
3 T
N
4 T
N
b FGF
5 T
N
6 T
N
T
7.0 Kb
28 S
Et Br staining
18 S
Fig, 1. bFGF mRNA expression in normal human kidney and renal cell carcinoma tissues, bFGF mRNA levels determined by Northern
analysis in renal cell carcinoma surgically removed tumor specimens (T) and corresponding normal tissues (N). Numbers above the lanes are sample numbers. The intensities of autoradiographic signals were determined by densitometry. Etidium bromide staining revealed that nearly equal amounts of RNA were present in each lane. 940
Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. 2. Immunohistochemical staining of bFGF in renal cell carcinoma tissue. Details of the experimental conditions were as
described in "Materials and Methods". The immunoreactivity is confined to the nucleus of tumor cells (arrow head). A: nonimmunized rabbit IgG; B: antibFGF rabbit IgG. Scale bar=10 I~m.
which is shown in Fig. 2, The results showed that there was a strong bFGF immunoreactivity at the nuclei of the tumor cells, and the cell surface was also positively stained. In normal renal tissue, little immunoreactivity was present at the glomerular cells and the tubular cells (data not shown). These findings suggest that increases of bFGF mRNA cause the expression of bFGF in renal cell carcinoma tissue, and that bFGF may regulate the tumor cell growth (nuclear FGF) or the neovascularization (cell surface FGF). 941
Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS DISCUSSION
The conversion of a normal cell to a malignant one is a multifactorial process (14). One of the important processes is deregulation of the growth factor and growth factor receptor gene expression.
For example, recent
studies of renal cell carcinoma suggest that overexpression of EGF receptor, TGF-c~ and TGF-131 genes may play an important role in either initiation or progression of malignant transformation (15,16). On the other hand, bFGF is known as a potent angiogenic factor and has been purified from various tissues, including kidney (17).
Shahabuddin et al. reported that tumor
angiogenesis factor (TAF) is present in renal cell carcinoma tissue (18). There are some angiogenic factors that have been already reported. Angiogenin has been isolated from the conditioned medium of a human colonic adenocarcinoma line (19), and Usuki et al. recently found that platelet-derived endothelial cell growth factor (PD-ECGF) is produced by a human squamous cell carcinoma cell line and two human anaplastic thyroid carcinoma cell lines (20).
However, angiogenin and PD-ECGF have not yet been identified in
renal Cell carcinoma tissue. In this study, we examined the expression of bFGF that might be related to the development of renal cell carcinoma. The bFGF mRNA transcripts of 7.0 kb were detected in all of the renal cell carcinoma examined (Fig. 1).
In
addition, the mRNA levels of bFGF in renal cell carcinoma were higher than corresponding normal renal tissue. To contribute to the elucidation of the role of bFGF
in the evolution
of renal cell carcinoma,
we performed
immunohistochemical staining of tumor tissue using anti-bFGF antibody, and found that immunoreactive bFGF was present in the nucleus and on the cell surface of the tumor cells (Fig. 2). This indicates that bFGF appears to relate to tumorigenesis of renal cell carcinoma.
Dell'Era et al. reported that
endogenous bFGF undergoes an intracellular sorting to the nucleus of the endothelial cell (21). We also present the immunohistochemical evidence on the presence of bFGF in the nucleus of renal cell carcinoma cells.
It may
suggest that nuclear FGF can promote the autocrine growth of tumor cells and 942
Vol. 183, No. 3, 1 9 9 2
BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS
cell surface FGF is able to stimulate the endothelial cell growth in a paracrine fashion.
However, there is no obvious explanation about a specific
mechanism of nuclear and cell surface FGF. Tanimoto et al. reported that bFGF mRNA expression in gastric carcinoma is higher than in corresponding normal gastric mucosa, especially, in scirrhus type (9).
All of the tumors used in our study showed
hypervascularity in the angiographic study and were low grade and low stage tumors, and no remarkable correlation between tumor character and clinical course was found. From our present data, it is suggested that bFGF may play an important role in progression of renal cell carcinoma, and bFGF may be the only TAF in renal cell carcinoma.
Further study of the signal transduction
pathway of bFGF in renal cell carcinoma is necessary in order to clarify the basic aspects of the biological character of human renal cell carcinoma.
ACKNOWLEDGMENTS We thank our branch hospital doctors for providing tissue samples and T. Shimogama, E. Taguchi, M. Yoshimoto, S. Honda and E. Suda for their technical assistance. This work was partially supported by the Grant-in-Aid for Scientific Research, the Ministry of Education, Science, and Culture, Japan.
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