oon1-9~~X/92/7506-1436503.00/0 .Journal of Clinical Endocrinology and Metabolism Copyright (0 1992 by The Endocrine Society
Vol. 75, No. 6 Printed in U.S A.
Identification and Characterization Weight Forms of Basic Fibroblast Human Pituitary Adenomas*
of High Molecular Growth Factor in
YAOHUA LI, MASAFUMI KOGA, NOR10 ARITA, TORU HAYAKAWA,
MATSUMOTO,
SOJI AND
Departments of Internal Medicine III (Y.L., M.K., (K.M.), Osaka University Hospital, Fukushima-ku,
KASAYAMA, KEISHI BUNZO SAT0 S.K., B.S.), Neurosurgery Osaka 553, Japan
(N.A., T.H.), and Pathology
ABSTRACT Basic fibroblast growth factor (bFGF) extracted from 23 human pituitary adenomas and 2 nontumorous anterior pituitary glands was examined by Western blot analysis to elucidate their molecular forms. Four bands that specifically interacted with anti-bFGF antibody were identified at the regions of 16K, 18K, 22K, and 24K mol wt. These immunodetectable bFGFs were bound to a heparin-Sepharose affinity column and eluted with 1-2 M NaCl. These partially purified tumor extracts could stimulate dose-dependently the growth of FGF-respon-
sive cells. Some tumors contained bFGF
T
factors would be expected to play an important role in the growth of human pituitary adenomas (18). Attempts have been directed to characterize the growth factors in human pituitary adenomas (19-21). Silverlight et al. (21) reported that bFGF levels in many human pituitary adenomas are lower than those in normal anterior pituitary gland, but the molecular forms of bFGF remain to be elucidated. Thus, the present study was designed to clarify the possible molecular heterogeneity of bFGF in human pituitary tumors and nontumorous anterior pituitary glands using Western blot analysis.
with a mol wt of 24 K as a dominant form. In contrast, a major form of bFGF at the region of 18K was identified in the nontumorous anterior pituitary glands. We also addressed the possible presence of FGF receptor mRNA using Northern blot analysis. Many, but not all, human pituitary adenomas were found to contain detectable levels of FGF receptor mRNA with a size of 3.5 kilobases. The present results suggest that the role of bFGF in growth and hormone secretion in human pituitary adenomas should be examined using high mol wt forms of bFGF. (J Clin Endocrinol Metab 75: 1436-1441, 1992)
HE NORMAL anterior pituitary gland possesses the ability to synthesize such growth factors as basic fibroblast growth factor (bFGF) (1, 2), epidermal growth factor (3), insulin-like growth factor-I (4), insulin-like growth factor-II (5), transforming growth factor-a (6), and nerve growth factor (7). In particular, the content of bFGF is thought to be high in pituitary cells (8). Although the effects of bFGF on the growth and PRL secretion of cultured pituitary cells have been examined (9-ll), its physiological role in pituitary cell function remains largely unknown. Characterization of cDNA encoding human bFGF showed that bFGF consists of 155 amino acids with a mol wt of 18,400 using AUG (methionine) as an initial codon (12). Recent observations using an in vitro translation system, however, showed that three CUG codons located up-stream of AUG can also be used as initial codons, and high mol wt forms (22K, 22.5K, and 24K) of bFGF are biosynthesized from a single mRNA (13). The NH2-terminal extended region present in a 24K form of bFGF has been demonstrated to contain the nuclear localization signal (14). Deletion of the signal from acidic FGF, which is structurally related to bFGF, has been found to markedly affect its function (15). Human pituitary adenomas have been suggested to arise as clonal expansion of genomically altered cells (16, 17), although the possibility is not eliminated that hyperplasia may precede clonal expansion in some tumors. Some growth
Subjects
and Methods
Patients Tumors from 23 patients undergoing transsphenoidal surgery for pituitary adenomas were studied. The tumors were identified by standard clinical, biochemical, and imaging criteria. Immunohistochemical analyses using antipituitary hormone antibodies were also carried out (22). Ten GH-producing, 12 clinically nonsecreting, and 1 ACTH-producing adenomas were included in this study. Two nontumorous pituitary glands used in the present study, which were resected to confirm total removal of adenomas, were obtained from patients with prolactinoma and ACTH-producing adenoma, respectively. Serum levels of pituitary hormones were measured by standard RIAs, as previously reported (23). Informed consent was obtained from all patients.
Extraction of bFGF from tissues and heparin-Sepharose affinity chromatography
Received November 14, 1991. Address requests for reprints to: Dr. Bunzo Sato, Department of Internal Medicine III, Osaka University Hospital, Fukushima-ku, Osaka 553, Japan. * This work was supported by grants from the Ministry of Education (Tokyo, Japan) and the Cancer Research Promotion Fund. t Sponsored by a Sasagawa Research Fellowship.
A sample of fresh tumor or nontumorous tissue was snap-frozen immediately after removal. Tumor tissue was also preserved for histological and immunohistochemical evaluations. The frozen tissue was maintained at -80 C for less than 1 month until used. The following procedures for tissue preparations were carried out at O-4 C. The frozen
1436
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bFGF IN PITUITARY tissue was thawed in 8 vol ice-cold homogenization buffer [lo mM TrisHCl, pH 7.4, at 20 C containing 1 M NaCl, 0.1% (wt/vol) 3-[(3cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS), 0.2 mg/mL leupeptin, and 1 mM phenylmethylsulfonylfluoride] (21). Homogenization was carried out in a Teflon-glass homogenizer (five 5-s bursts at 1,000 ‘pm, each burst was followed by 20 s of cooling at 0 C). The homogenate was centrifuged at 105,000 X g for 60 min. The supernatant was diluted with an equal volume of 10 mM Tris-HCl, pH 7.4, at 20 C. This diluted supernatant (usually 3 mL) was applied to heparin-Sepharose (gel bed volume, 0.1 mL) that had been preequilibrated with 10 mM Tris-HCl, pH 7.4, at 20 C containing 0.5 M NaCl and 0.1% (wt/vol) CHAPS. The bFGF thus obtained was washed with 20 mL preequilibrated buffer and eluted with 2 mL 2 M NaCl in 10 mM Tris-HCl, pH 7.4, at 20 C and 0.1% (wt/vol) CHAPS, unless stated otherwise. Western blot analyiis After the addition of 100 rg insulin as a carrier protein, 2 M NaCl eluate from the heparin-Sepharose affinity column was mixed with 100% trichloroacetic acid (TCA) fo adjust the final TCA concentration to 10%. This solution was left to stand at 0 C for 15 h, and the precipitate was collected by centrifugation at 10,000 X g for 10 min and washed twice with 10% TCA and twice with ethanol-ethylether (l:l, vol/vol). The washed precipitate was dissolved in 6-12 ILL sodium dodecyl sulfate (SDS) sample buffer (24) and boiled for 5 min. An aliquot (1-3 pL) of samples was electrophoresed on Phast Gel Homogenous 20 (Pharmacia, Uppsala, Sweden) and transferred to the nitrocellulose membrane using Phast System Separation and Control Unit (Pharmacia). To calculate the molecular size, prestained markers were used. Human bFGF, obtained from R & D Systems (Minneapolis, MN), was simultaneously electrophoresed. According to the manufacturer, this bFGF consists of 146 amino acids, and has a mol wt of 16K (NHz-terminal deleted form). The material was blotted, and the nitrocellulose membrane was treated with 5 mg/mL gelatin in PBS for 30 min at 37 C and incubated with antibFGF antibody (l:l,OOO diluted with the above buffer) for 15 h at 4 C. Two antibodies used here, kindly supplied by Dr. M. Klagsbrun, recognized the NH*-terminal (R30) and mid (R4/6) portions of bovine bFGF, respectively (25). The proteins recognized by these antibodies were visualized by successive incubations with avidin-biotin complex reagents according to the manufacture’s instruction (Vector Laboratories, Burlingame, CA). Semiquantitative analyses were carried out by densitometric scanning, as reported previously (26). Northern
ADENOMAS Materials
All tissue culture materials were purchased from Corning Glass Works (Corning, NY). Minimum Essential Medium and Ham’s F-12 were obtained from Nissui Pharmaceutical Co. Ltd (Tokyo, Japan). Fetal bovine serum was purchased from HyClone Laboratories, Inc. (Logan, UT). Heparin-Sepharose was obtained from Pharmacia. Ampure SA and DT we;e obtained from Amersham Japan (Tokyo, Japan).-[MethyI-3H]thymidine (SA. 70-90 Ci/mmol) and 13’Pldeoxv-CTP (SA. 3000 Ci/mmol) were purchased from New’ England’Nuclear (Cambridge, MA). The other reagents used here were of analytical grade.
Results Demonstration nontumorous
of bFGF in human pituitary anterior pituitary gland
adenomas
and
Western blot analyses revealed that the antibody (R30) against the NHz-terminal portion of bovine bFGF reacted with authentic 16K human bFGF. Preincubation of antibody with 10 pg/ml human bFGF resulted in the disappearance of the 16K band (Fig. 1A). Similar experiments were carried out using the extract from nonsecreting adenoma. By using the eluate from the heparin-Sepharose column with 2 M NaCl, several bands could be visualized by Western blot analysis (Fig. 1B). The results further obtained by the use of antibody preincubated with human bFGF or preimmune serum showed that anti-bFGF antibody specifically reacted with 18K (doublet), 22K, and 24K polypeptides. Analysis of the extract from nontumorous anterior pituitary gland showed that both antibodies against NH*-terminal (R30) and mid (R4/6) portions of bovine bFGF recognized 18K (doublet) and 24K proteins (Fig. 1C). The intensity of the 24K band was much lower than that of the 18K band in the nontumorous anterior pituitary gland extract (see below). Since R4/6 recognized more nonspecific bands and showed lower
C
B bFGF 7
PRE
k
R30 R416
blot analysis
Total RNA was extracted with guanidium isothiocyanate (27) and electrophoresed on a 1% agarose gel containing 0.66 M formaldehyde (10 pg/lane). After transferal to a nitrocellulose filter, Northern blot analysis was performed as described previously (26). The probes used here were the SalI-ApaI fragment [0.4 kilobase (kb)], encoding the region from the juxtamembrane to the kinase domain of chick FGF receptor, which was kindly supplied by Dr. L. T. Williams (28), and the HinfIHinfI fragment (0.4 kb), encoding the mouse fl-actin (Wako Pure Chemicals, Osaka, Japan).
bFGF Mitogenic
1437
activity
Mitogenic activity in the eluate from the heparin-Sepharose affinity column was assessed by [3H]thymidine incorporation into the target cell line [SC-3 cells established from mouse mammary carcinoma (29)], as described previously (30), after CHAPS and NaCl were removed by successive passages of the eluate through Ampure DT and SA columns (31). This cell line was chosen as the target cell because the growth of SC-3 cells is known to be stimulated in a FGF- or FGF-related growth factor-specific manner (30). After 24-h stimulation with test compounds in the serum-free medium, the cells were pulse labeled with 0.15 PCi [3H]thymidine/well for 2 h, as described previously (30).
Anterior Pituitary
FIG. 1. Demonstration of immunoreactive bFGF in nonsecreting pituitary adenoma or nontumorous anterior pituitary gland by Western blot analysis. Extract from nonsecreting pituitary adenoma (B) or nontumorous anterior pituitary gland (C) was partially purified on a heparin-Sepharose column and electrophoresed. Authentic bFGF (0.5 ng) was also electrophoresed (A). After transfer to nitrocellulose membrane, the membranes were incubated with antibodies untreated (bFGF -) or pretreated with 10 Fg/mL bFGF at 4 C for 18 h (bFGF +). Preimmune serum (PRE) was also used. The arrows indicate the bands specifically recognized by anti-bFGF antibodies. The positions of the markers for determining mol wt are also depicted.
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1438
LI ET AL.
sensitivity than R30 (Fig. lC), the following experiments were carried out using R30. The elution profile of the immunodetectable bFGF from the heparin-Sepharose affinity column was also examined (Fig. 2). No specific bands could be detected by elution with 1.0 M NaCl, but elution with 2.0 M NaCl gave immunodetectable bFGF from the heparinSepharosecolumn that had been prewashed with 1.0 M NaCl. Again, the high mol wt form of bFGF (24K) was identified as the dominant form in human pituitary adenomas (Fig. 2, lane 5). Next, the molecular forms of bFGF in various pituitary tissues were addressed (Fig. 3). All adenomas (GHproducing, nonsecreting and ACTH-producing adenomas) examined in this experiment were found to contain bFGF with a mol wt of 24K, and one GH-producing tumor showed an additional dense band at the region of 18K (Fig. 3, lane 2). Furthermore, a major form of immunodetectable bFGF in nontumorous anterior pituitary tissues was identified as an
__ *
32.5 27.5 -
JCE & M. 1992 Vol75.No6
18K polypeptide doublet with an additional faint band at 24K region (Fig. 3, lane 6). Mitogenic
activity of human pituitary
adenoma-derived
bFGF
The mitogenic activity in 2 M NaCl extract from human pituitary adenoma was measured(Fig. 4). Two of the extracts used here remarkedly enhanced the DNA synthesis of SC-3 cells in a dose-dependent manner which was in parallel to that elicited by authentic human bFGF. All five adenomas examined showed marked growth-promoting activity (data not shown). The effects of anti-bFGF neutralizing antibody on the growth-promoting activity also were examined using three of the tumor extracts (Fig. 4B). These activities were reduced almost to nonstimulation levels by the addition of
i
0
.OOl
18.5 -
r x1ooo0
.Ol bFGF XlWO
.l
1
10
Xl00
xl0
1 Xl
(nglml)
Dilution FIG. 2. Immunodetectable bFGF in Western blots of heparin-sepharose affinity column eluates of nontumorous or tumorous pituitary extracts. Extract from a nontumorous anterior pituitary gland (lanes 2 and 3) or a GH-producing adenoma (lanes 4 and 5) was diluted to reduce its NaCl concentration to 0.5 M and then applied to a heparinSepharose affinity column. After washing, the materials bound to the column were eluted with 1.0 M NaCl (lanes 2 and 4) and further eluted with 2.0 M NaCl (lanes 3 and 5). These samples as well as authentic bFGF (2 ng; lane 1) were analyzed by the Western blot procedure.
B
123456
Case
FIG. 3. Molecular heterogeneity of bFGF in various pituitary tissues. The mol wt of immunodetectable bFGF in GH-producing (lanes 2 and 4), nonsecreting (lane 3), and ACTH-producing (lane 5) tumors were determined by Western blot analysis. The authentic bFGF (0.5 ng; lane 1) and nontumorous anterior pituitary gland extract (lane 6) were also analyzed.
:I1 no.
6
21
17
bFGF
FIG. 4. Mitogenic activity of FGF-responsive SC-3 cells in pituitary tumor extracts. A, Two pituitary tumor extracts (0 and A) were partially purified by successive passing through heparin-Sepharose, Ampure SA, and Ampure DT columns, and finally eluted with an initial volume of the serum-free medium. After dilution with various volumes of the serum-free medium, their mitogenic activity was measured, as described in Muteriak and Methods. The mitogenic activity of various concentrations of authentic bFGF was also assayed (0). B, Three tumor extracts (cases 6, 21, and 17; see Table 1) were treated as described above. Nondiluted samples were incubated with SC-3 cells in the absence (0) or presence (B) of 50 pg/mL anti-bFGF neutralizing antibody (R & D Systems). SC-3 cells were also stimulated with 0.1 ng/mL bFGF. Their mitogenic activity was measured. All assays were carried out in triplicate. The SES were not depicted because their values were always less than 10%.
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bFGF IN PITUITARY 50 &ml anti-bFGF neutralizing antibody (R & D System). Control rabbit immunoglobulin G failed to inhibit the growth-promoting activity (data not shown). These studies further indicated that human pituitary adenoma can synthesize biologically active bFGF. Northern
blot analysis of FGF receptor
mRNA
and biochemical
analyses in each adenoma
The data on immunodetectable bFGF and FGF receptor mRNA are summarized in Table 1. Clinical profiles also are provided. Immunoreactive bFGF could not be detected in only one nonsecreting adenoma. Four of 10 GH-producing adenomas and 2 of 12 nonsecreting adenomas contained large amounts of 24K bFGF, and 1 ACTH-producing adenoma contained only 24K bFGF. Two nontumorous anterior pituitary glands contained 18K bFGF as a dominant form, with a small quantity of 24K form. We also detected 16K bFGF. This was probably the product of degradation of the higher mol wt forms of bFGF (13). Although qualitative, rather than quantitative, results could be obtained by Western blot analysis, densitometric scanning revealed that some
Caseno.
1 14 13
2
10
11 23
3 16 12 5
FGF Receptor 28s 3.5kb
*
18s
*
p-actin 28s
1439
tumors contained levels of bFGF, similar to those in nontumorous anterior pituitary. Three of 11 adenomas did not possess detectable levels of FGF receptor mRNA. Interestingly, the FGF receptor mRNA level in bFGF-negative nonsecreting adenoma (case 12) was below detectable limits. Discussion
In discussing the biological role of bFGF, the possible presenceof FGF receptor in human pituitary adenomas must not be overlooked. Antibodies to FGF receptor protein, however, were not available, and we had to rely a Northern blot technique in our analysis of FGF receptor mRNA. As shown in Fig. 5, the band hybridizable with chick FGF receptor cDNA was identified at the 3.5 kb region in many, but not all, adenomas. Its size (3.5 kb) was similar to that reported in chick (28), mouse (26), and human (32) cells. Since a relatively large amount of tissue is needed to analyze FGF receptor mRNA, only a limited number of adenomas could be studied. Nonetheless, the results suggest that many adenomas contain FGF receptor. Clinical
ADENOMAS
-
FIG. 5. Northern blot analyses of FGF receptor mRNA in pituitary adenomas. Total RNA from various pituitary adenomas was extracted and electrophoresed. Northern blot analyses were carried out using FGF receptor (upper panel) or P-actin (lower panel) cDNA. These bands were visualized by autoradiography at -80 C for 5 days (FGF receptor) or 18 h (@actin). The positions of 18s and 28s ribosomal RNAs were also depicted. The samples used here were identified by patient case number (see Table 1).
The present studies have clearly demonstrated that human pituitary adenomas contain bFGF. This was confirmed by the following observations. 1) Heparin-binding moleculesin tumor extracts specifically cross-reacted with anti-bFGF antibodies when analyzed by electrophoretic blotting. 2) These immunoreactive molecules were eluted at 1.0-2.0 M NaCl from a heparin-Sepharose affinity column. 3) They were potent mitogens for FGF-responsive SC-3 cells. In addition, the antibodies used here have been reported to be relatively specific for bFGF and not to be reactive to acidic FGF (25); moreover, a certain amount of nonspecific background reaction was observed after immunoblotting, probably because the absolute amount of bFGF in the pituitary tissues was relatively low (33). The major form of bFGF present in the extract from nontumorous anterior pituitary glands was 18K, although we examined only two nontumorous pituitary glands. It is extremely difficult to obtain nontumorous pituitary glands. In addition, postmorten degradation of bFGF molecules(data not shown) prevented us from using autopsy specimens.On the other hand, some human pituitary adenomashave been observed to produce predominantly the high mol wt form (24K) of bFGF. Recently, such high molecular forms (2225K) have been demonstrated in various tissuesor cells (3336). These appear to be the NH2-terminal extended form, derived from a single bFGF mRNA via the use of three CUD initial codons (13, 37). We could identify only two high mol wt forms of bFGF in our human pituitary tissues,probably becausethe mol wt of the two bFGF species(22K and 22.5K) were too similar to obtain sufficient separation. It is still not known whether the NHz-terminus initiates at an AUG codon (18K) or at one of the CUG codons (22K and 24K). Artificial degradation of bFGF from 24K to 18K has been suggestedto occur during the storage of tissuesat -20 C (34). The high mol wt form of guinea pig bFGF can also be converted to an 18K form by trypsin (38). On the other hand, Renko et al. (33) have demonstrated that the control of tryptic activity is not so critical to the distribution of three bFGF forms. While the contribution of protease activities to the molecular heterogeneity of bFGF has not been rigorously eliminated, it should be pointed out that the simultaneously processed adenomas and nontumorous pituitary anterior glands showed clear differences in the mol wt of their bFGF. Our Western blot procedure used here was qualitative, but not quantitative, since the recovery of bFGF was relatively poor (-10%). Also, our data did not demonstrate without doubt that bFGF is present in the adenoma cells as it is in other cell types, such as endothelium, and folliculostellate cells. Even if nontumorous cells are synthesizing bFGF, however, the adenoma cellsmay be exposed to bFGF, sincebFGF is known to adhere to the extracellular matrix (39). In addi-
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TABLE glands
JCE & M - 1992 Vol75.No6
LI ET AL.
1440 1. Characteristics
Case
of immunoreactive
no.
GH-producing adenomas 1 2 3 4 5 6 7 8 9 10 Nonsecreting adenomas 11 12 13 14 15 16 17 18 19 20 21 22 ACTH-producing adenomas 23 Normal anterior pituitary glands 24 25 “The contents blot and classified
bFGF
and FGF
receptor
Tumor (diameter,
Age (yd
Sex
51 38 44 42 30 47 32 49 45 70
M F ii M M M M M M
15 8 15 12 32 24 20 NA 30
58 42 58 67 33 63 68 33 35 39 71 58
M F F M F F F M M M M F
30 18 25 15 40 20 20 NA NA NA NA NA
39
F
13
size mm)
10
mRNA
in human
pituitary
Immunoreactive 16K
18K
adenomas
and normal
bFGF 22K
+++ -
++ ++ +++ ++
pituitary tissues were semiquantified from the densitometric group. ND, Not determined; NA, data were not available.
tion, the FGF receptor is obligatory in eliciting FGF activities. To our knowledge, this is the first report to demonstrate the presence of FGF receptor mRNA in human pituitary adenoma. Interestingly, our bFGF-negative tumors did not contain detectable levels of FGF receptor mRNA. In another study we demonstrated that bFGF can up-regulate FGF receptor mRNA in SC-3 cells (26). These findings tempt us to speculate that the lack of bFGF synthesis arrests FGF receptor mRNA expression. The biological role of bFGF in the growth and function of normal or transformed pituitary cells has been mainly examined by NH2-terminal deleted forms (16K and 18K) of bFGF in the culture medium. However, a recent finding has suggested the existence of different signal pathways for bFGF, depending on whether bFGF is produced by or provided to the cells (40). Moreover, the NH2-terminus present in a 24K form of bFGF has been proven to contain the nuclear localization signal (14, 33). The subcellular and extracellular localization of bFGF in the development and progression of pituitary adenoma should be examined in future studies. Acknowledgments The authors acknowledge Prof. T. Kishimoto for his encouragement for the present study. We also thank our clinical staff for taking care of the patients and Mr. D. Elick for his editorial help.
pituitary
FGF receptor mRNA
24K
+++ ++ ++
39 38
of each mol wt form of bFGF in human +, ++, and +++ in the bFGF-positive
anterior
+++ ++
+++ +++ + +++ ++ ++
ND ++ ND ND ND ND -
++ +++ ++ +++ ++ -
+++ ++ ++ ND ++ ND ND ND ND ND ND
+++
++
+ ++
ND ND scanning
data on Western
References 1. Gospodarowicz D. 1974 Localization of a fibroblast growth factor and its effect alone and with hydrocortisone on 3T3 cell growth. Nature. 249:123. 2. Esch F, Baird A, Ling N, et al. 1985 Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Proc Nat1 Acad Sci USA. 82:6507-6511, 3. Kasselberg AG, Orth DN, Gray ME, Stablman MT. 1985 Immunocytochemical localization of human epidermal growth factor/ urogastrone in several human tissues. J Histochem Cytochem. 33:315-322. 4. Binoux M., Hossenlopp P, Lassarre C, Hardouin N. 1981 Production of insulin-like growth factors and their carrier by rat pituitary gland and brain explants in culture. FEBS Lett. 124:178-184. 5. Hasselbacher GK, Schwab ME, Psai A, Humbel RE. 1985 Insulinlike growth factor II (IGF-II) in human brain: region distribution of IGF-II and higher molecular mass forms. Proc Nat1 Acad Sci USA. 82:2153-2157. 6. Kobrin MS, Asa SL, Samsoondar J, Kudlow JE. 1987 a-Transforming growth factor in the bovine anterior pituitary gland: secretion by dispersed cells and immunohistochemical localization. Endocrinology. 121:1412-1416. 7. Lahtinen T, Soinila S, Lakshmanan J. 1989 Biological demonstration of nerve growth factor in the rat pituitary gland. Neuroscience. 30:165-170. 8. Gospodarowicz D. 1975 Purification of a fibroblast growth factor from bovine pituitary. J Biol Chem. 250:2515-2520. 9. Schonbrunn B, Krasnoff M, Westendonf JM, Tashjiian AH. 1980 Epidermal growth factor and thyrotropin-releasing hormone act similarly on clonal pituitary cell strain. J Cell Biol. 85:786-797.
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bFGF
IN PITUITARY
10. Baird A, Mormede P, Ying S-Y, et al. 1985 A nonmitogenic pituitary function of fibroblast growth factor: regulation of thyrotropin and prolactin secretion. Proc Nat1 Acad Sci USA. 82:5545-5549. 11. Mormede P, Baird A. 1988 Estrogen, cyclic adenosine 3’,5’-monophosphate, and phorbol esters modulate the prolactin response of GH, cells to basic fibroblast growth factor. Endocrinology. 12212265-2271. 12. Abraham JA, Whang JL, Tumolo A, Mergia A, et al. 1986 Human basic fibroblast growth factor: nucleotide sequence and genomic organization. EMBO J. 5:2523-2528. 13. Florkiewicz RZ, Sommer A. 1989 Human basic fibroblast growth factor gene encodes four polypeptides: three initiate translation from non-AUG codons. Proc Nat1 Acad Sci USA. 86:3978-3981. 14. Quart0 N, Finger FP, Rifkin DB. 1991 The NHz-terminal extension of high molecular weight basic fibroblast growth factor is a nuclear targeting signal. J Cell Physiol. 147:311-318. 15. Imamura T, Engleka K, Zhan X, et al. 1990 Recovery of mitogenic activity of a growth factor mutant with a nuclear translocation sequence. Science. 249:1567-1570. 16. Herman V, Fagin J, Gonsky R, Kovacs K, Melmed S. 1990 Clonal origin of pituitary adenomas. J Clin Endocrinol Metab. 71:14271433. 17. Alexander JM, Biller BM, Bikkal H, Zervas NT, Arnold A, Klibanski A. 1990 Clinically nonfunctioning pituitary tumors are monoclonal in origin. J Clin Invest. 86:336-340. 18. Cross M, Dexter TM. 1991 Growth factors in development, transformation, and tumorigenesis. Cell. 64:271-280. 19. Kaspar S, Friesen HG. 1986 Human pituitary tissue secretes a potent growth factor for chondrocyte proliferation. J Clin Endocrinol Metab. 62:70-76. J, Ham J, Bevan JS, ten Horn CD, Scanlon MF. 1991 20. Webster Preliminary characterization of growth factors secreted by human pituitary tumors. J Clin Endocrinol Metab. 72:687-692. JJ, Prysor-Jones RA, Jenkins JS. 1990 Basic fibroblast 21. Silverlight growth factor in human pituitary tumors. Clin Endocrinol (Oxf). 321669-676. 22. Mori H, Mori S, Saitoh Y, et al. 1985 Effect of bromocriptine on prolactin-secreting pituitary adenoma. Cancer. 56:230-238. 23. Koga M, Nakao H, Arao M, et al. 1987 Demonstration of specific dopamine receptors on human pituitary adenomas. Acta Endocrinol (Copenh). 114:595-602. 24. Laemmli UK. 1970 Cleavage of structural proteins during the assembIy of the head of bacteriophage T+ Nature. 227:680-685. 25. Klagsbrun M, Sasse J, Sullivan R, Smith JA. 1986 Human tumor cells synthesize an endothelial cell growth factor that is structurally related to basic fibroblast erowth factor. Proc Nat1 Acad Sci USA. 83:2448-2452. 26. Saito H, Kasayama S, Kouhara H, Matsumoto K, Sato B. 1991 Upregulation of fibroblast growth factor (FGF) receptor mRNA levels by basic FGF or testosterone in androgen-sensitive mouse
ADENOMAS
27.
28.
29.
30
31
32
33
34
35
36.
37.
38.
39. 40.
1441
mammary tumor cells. Biochem Biophys Res Commun. 174:136141. Chirgwin J, Pryzbyla A, McDonald R, Rutter W. 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 18:5294-5299. Lee PL, Johnson DE, Cousens LS, Fried VA, Williams LT. 1989 Purification and complementary DNA cloning of a receptor for basic fibroblast growth factor. Science. 245:57-60.Noeuchi S, Nishizawa Y, Nakamura N, et al. 1987 Growthstimulating effect of pharmacological doses of estrogen on androgen-dependent Shionogi carcinoma 115 in vivo but not in cell culture. Cancer Res. 47:263-268. Nakamura N, Yamanishi H, Lu J, et al. 1989 Growth-stimulatory effects of androgen and high concentration of glucocorticoid or fibroblast growth factors on a cloned cell line from Shionogi carcinoma 115 cells in a serum-free medium. J Steroid Biochem. 33:1318. Kasayama S, Sumitani S, Matsumoto K, Sato B. 1991 Heparin inhibits autocrine stimulation but not fibroblast growth factor stimulation of cell proliferation of androgen-responsive Shionogi carcinoma 115. J Cell I’hysiol. 148:260-266. Houssaint E, Blanquet PR, Champion-Aranaud P, et al. 1990 Related fibroblast growth factor receptor genes exist in the human eenome Proc Nat1 Acad Sci USA. 87:8180-8184. Renko M, Quart0 N, Morimoto T, Rifkin DB. 1990 Nuclear and cytoplasmic localization of different basic fibroblast growth factor species. J Cell Physiol. 144:108-114. Presta M, Statuto M, Rusnati M, Dell’Era P, Ragnotti G. 1989 Characterization of a Mr 25,000 basic fibroblast growth factor form in adult, regenerating, and fetal rat liver. Biochem Biophys Res Commun. 164:1182-1189. Sommer A, Moscatelli D, Rifkin DB. 1989 An amino-terminally extended and post-translationally modified form of a 25 Kd basic fibroblast growth factor. Biochem Biophys Res Commun. 160:12671274. Maier JAM, Rusnati M, Ragnotti G, Presta M. 1990 Characterization of a Mr 20,000 basic fibroblast growth factor-like protein secreted by normal and transformed fetal bovine aortic endothelial cells, Exp Cell Res. 186:354-361. Prats H, Kaghad H, Prats AC, et al. 1989 High molecular mass forms of ba& fibroblast growth factor are initiated by alternative CUG codons. Proc Nat1 Acad Sci USA. 86:1836-1840. Moscatelli D, Joseph-Silverstein J, Manejias R, Rifkin DB. 1987 Mr 25,000 heparin:binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor. Proc Nat1 Acad Sci USAu84:5778-5782. Rifkin DB, Moscatelli D. 1989 Recent developments in the cell biology of basic fibroblast growth factor. J Cell Biol. 109:1-6. Burgess WH, Maciag T. 1989 The heparin-binding (fibroblast) family of proteins. Annu Rev Biochem. 58:575-606.
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Vol. 75, No. 6 Printed in U.S A.
Identification and Characterization Weight Forms of Basic Fibroblast Human Pituitary Adenomas*
of High Molecular Growth Factor in
YAOHUA LI, MASAFUMI KOGA, NOR10 ARITA, TORU HAYAKAWA,
MATSUMOTO,
SOJI AND
Departments of Internal Medicine III (Y.L., M.K., (K.M.), Osaka University Hospital, Fukushima-ku,
KASAYAMA, KEISHI BUNZO SAT0 S.K., B.S.), Neurosurgery Osaka 553, Japan
(N.A., T.H.), and Pathology
ABSTRACT Basic fibroblast growth factor (bFGF) extracted from 23 human pituitary adenomas and 2 nontumorous anterior pituitary glands was examined by Western blot analysis to elucidate their molecular forms. Four bands that specifically interacted with anti-bFGF antibody were identified at the regions of 16K, 18K, 22K, and 24K mol wt. These immunodetectable bFGFs were bound to a heparin-Sepharose affinity column and eluted with 1-2 M NaCl. These partially purified tumor extracts could stimulate dose-dependently the growth of FGF-respon-
sive cells. Some tumors contained bFGF
T
factors would be expected to play an important role in the growth of human pituitary adenomas (18). Attempts have been directed to characterize the growth factors in human pituitary adenomas (19-21). Silverlight et al. (21) reported that bFGF levels in many human pituitary adenomas are lower than those in normal anterior pituitary gland, but the molecular forms of bFGF remain to be elucidated. Thus, the present study was designed to clarify the possible molecular heterogeneity of bFGF in human pituitary tumors and nontumorous anterior pituitary glands using Western blot analysis.
with a mol wt of 24 K as a dominant form. In contrast, a major form of bFGF at the region of 18K was identified in the nontumorous anterior pituitary glands. We also addressed the possible presence of FGF receptor mRNA using Northern blot analysis. Many, but not all, human pituitary adenomas were found to contain detectable levels of FGF receptor mRNA with a size of 3.5 kilobases. The present results suggest that the role of bFGF in growth and hormone secretion in human pituitary adenomas should be examined using high mol wt forms of bFGF. (J Clin Endocrinol Metab 75: 1436-1441, 1992)
HE NORMAL anterior pituitary gland possesses the ability to synthesize such growth factors as basic fibroblast growth factor (bFGF) (1, 2), epidermal growth factor (3), insulin-like growth factor-I (4), insulin-like growth factor-II (5), transforming growth factor-a (6), and nerve growth factor (7). In particular, the content of bFGF is thought to be high in pituitary cells (8). Although the effects of bFGF on the growth and PRL secretion of cultured pituitary cells have been examined (9-ll), its physiological role in pituitary cell function remains largely unknown. Characterization of cDNA encoding human bFGF showed that bFGF consists of 155 amino acids with a mol wt of 18,400 using AUG (methionine) as an initial codon (12). Recent observations using an in vitro translation system, however, showed that three CUG codons located up-stream of AUG can also be used as initial codons, and high mol wt forms (22K, 22.5K, and 24K) of bFGF are biosynthesized from a single mRNA (13). The NH2-terminal extended region present in a 24K form of bFGF has been demonstrated to contain the nuclear localization signal (14). Deletion of the signal from acidic FGF, which is structurally related to bFGF, has been found to markedly affect its function (15). Human pituitary adenomas have been suggested to arise as clonal expansion of genomically altered cells (16, 17), although the possibility is not eliminated that hyperplasia may precede clonal expansion in some tumors. Some growth
Subjects
and Methods
Patients Tumors from 23 patients undergoing transsphenoidal surgery for pituitary adenomas were studied. The tumors were identified by standard clinical, biochemical, and imaging criteria. Immunohistochemical analyses using antipituitary hormone antibodies were also carried out (22). Ten GH-producing, 12 clinically nonsecreting, and 1 ACTH-producing adenomas were included in this study. Two nontumorous pituitary glands used in the present study, which were resected to confirm total removal of adenomas, were obtained from patients with prolactinoma and ACTH-producing adenoma, respectively. Serum levels of pituitary hormones were measured by standard RIAs, as previously reported (23). Informed consent was obtained from all patients.
Extraction of bFGF from tissues and heparin-Sepharose affinity chromatography
Received November 14, 1991. Address requests for reprints to: Dr. Bunzo Sato, Department of Internal Medicine III, Osaka University Hospital, Fukushima-ku, Osaka 553, Japan. * This work was supported by grants from the Ministry of Education (Tokyo, Japan) and the Cancer Research Promotion Fund. t Sponsored by a Sasagawa Research Fellowship.
A sample of fresh tumor or nontumorous tissue was snap-frozen immediately after removal. Tumor tissue was also preserved for histological and immunohistochemical evaluations. The frozen tissue was maintained at -80 C for less than 1 month until used. The following procedures for tissue preparations were carried out at O-4 C. The frozen
1436
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bFGF IN PITUITARY tissue was thawed in 8 vol ice-cold homogenization buffer [lo mM TrisHCl, pH 7.4, at 20 C containing 1 M NaCl, 0.1% (wt/vol) 3-[(3cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS), 0.2 mg/mL leupeptin, and 1 mM phenylmethylsulfonylfluoride] (21). Homogenization was carried out in a Teflon-glass homogenizer (five 5-s bursts at 1,000 ‘pm, each burst was followed by 20 s of cooling at 0 C). The homogenate was centrifuged at 105,000 X g for 60 min. The supernatant was diluted with an equal volume of 10 mM Tris-HCl, pH 7.4, at 20 C. This diluted supernatant (usually 3 mL) was applied to heparin-Sepharose (gel bed volume, 0.1 mL) that had been preequilibrated with 10 mM Tris-HCl, pH 7.4, at 20 C containing 0.5 M NaCl and 0.1% (wt/vol) CHAPS. The bFGF thus obtained was washed with 20 mL preequilibrated buffer and eluted with 2 mL 2 M NaCl in 10 mM Tris-HCl, pH 7.4, at 20 C and 0.1% (wt/vol) CHAPS, unless stated otherwise. Western blot analyiis After the addition of 100 rg insulin as a carrier protein, 2 M NaCl eluate from the heparin-Sepharose affinity column was mixed with 100% trichloroacetic acid (TCA) fo adjust the final TCA concentration to 10%. This solution was left to stand at 0 C for 15 h, and the precipitate was collected by centrifugation at 10,000 X g for 10 min and washed twice with 10% TCA and twice with ethanol-ethylether (l:l, vol/vol). The washed precipitate was dissolved in 6-12 ILL sodium dodecyl sulfate (SDS) sample buffer (24) and boiled for 5 min. An aliquot (1-3 pL) of samples was electrophoresed on Phast Gel Homogenous 20 (Pharmacia, Uppsala, Sweden) and transferred to the nitrocellulose membrane using Phast System Separation and Control Unit (Pharmacia). To calculate the molecular size, prestained markers were used. Human bFGF, obtained from R & D Systems (Minneapolis, MN), was simultaneously electrophoresed. According to the manufacturer, this bFGF consists of 146 amino acids, and has a mol wt of 16K (NHz-terminal deleted form). The material was blotted, and the nitrocellulose membrane was treated with 5 mg/mL gelatin in PBS for 30 min at 37 C and incubated with antibFGF antibody (l:l,OOO diluted with the above buffer) for 15 h at 4 C. Two antibodies used here, kindly supplied by Dr. M. Klagsbrun, recognized the NH*-terminal (R30) and mid (R4/6) portions of bovine bFGF, respectively (25). The proteins recognized by these antibodies were visualized by successive incubations with avidin-biotin complex reagents according to the manufacture’s instruction (Vector Laboratories, Burlingame, CA). Semiquantitative analyses were carried out by densitometric scanning, as reported previously (26). Northern
ADENOMAS Materials
All tissue culture materials were purchased from Corning Glass Works (Corning, NY). Minimum Essential Medium and Ham’s F-12 were obtained from Nissui Pharmaceutical Co. Ltd (Tokyo, Japan). Fetal bovine serum was purchased from HyClone Laboratories, Inc. (Logan, UT). Heparin-Sepharose was obtained from Pharmacia. Ampure SA and DT we;e obtained from Amersham Japan (Tokyo, Japan).-[MethyI-3H]thymidine (SA. 70-90 Ci/mmol) and 13’Pldeoxv-CTP (SA. 3000 Ci/mmol) were purchased from New’ England’Nuclear (Cambridge, MA). The other reagents used here were of analytical grade.
Results Demonstration nontumorous
of bFGF in human pituitary anterior pituitary gland
adenomas
and
Western blot analyses revealed that the antibody (R30) against the NHz-terminal portion of bovine bFGF reacted with authentic 16K human bFGF. Preincubation of antibody with 10 pg/ml human bFGF resulted in the disappearance of the 16K band (Fig. 1A). Similar experiments were carried out using the extract from nonsecreting adenoma. By using the eluate from the heparin-Sepharose column with 2 M NaCl, several bands could be visualized by Western blot analysis (Fig. 1B). The results further obtained by the use of antibody preincubated with human bFGF or preimmune serum showed that anti-bFGF antibody specifically reacted with 18K (doublet), 22K, and 24K polypeptides. Analysis of the extract from nontumorous anterior pituitary gland showed that both antibodies against NH*-terminal (R30) and mid (R4/6) portions of bovine bFGF recognized 18K (doublet) and 24K proteins (Fig. 1C). The intensity of the 24K band was much lower than that of the 18K band in the nontumorous anterior pituitary gland extract (see below). Since R4/6 recognized more nonspecific bands and showed lower
C
B bFGF 7
PRE
k
R30 R416
blot analysis
Total RNA was extracted with guanidium isothiocyanate (27) and electrophoresed on a 1% agarose gel containing 0.66 M formaldehyde (10 pg/lane). After transferal to a nitrocellulose filter, Northern blot analysis was performed as described previously (26). The probes used here were the SalI-ApaI fragment [0.4 kilobase (kb)], encoding the region from the juxtamembrane to the kinase domain of chick FGF receptor, which was kindly supplied by Dr. L. T. Williams (28), and the HinfIHinfI fragment (0.4 kb), encoding the mouse fl-actin (Wako Pure Chemicals, Osaka, Japan).
bFGF Mitogenic
1437
activity
Mitogenic activity in the eluate from the heparin-Sepharose affinity column was assessed by [3H]thymidine incorporation into the target cell line [SC-3 cells established from mouse mammary carcinoma (29)], as described previously (30), after CHAPS and NaCl were removed by successive passages of the eluate through Ampure DT and SA columns (31). This cell line was chosen as the target cell because the growth of SC-3 cells is known to be stimulated in a FGF- or FGF-related growth factor-specific manner (30). After 24-h stimulation with test compounds in the serum-free medium, the cells were pulse labeled with 0.15 PCi [3H]thymidine/well for 2 h, as described previously (30).
Anterior Pituitary
FIG. 1. Demonstration of immunoreactive bFGF in nonsecreting pituitary adenoma or nontumorous anterior pituitary gland by Western blot analysis. Extract from nonsecreting pituitary adenoma (B) or nontumorous anterior pituitary gland (C) was partially purified on a heparin-Sepharose column and electrophoresed. Authentic bFGF (0.5 ng) was also electrophoresed (A). After transfer to nitrocellulose membrane, the membranes were incubated with antibodies untreated (bFGF -) or pretreated with 10 Fg/mL bFGF at 4 C for 18 h (bFGF +). Preimmune serum (PRE) was also used. The arrows indicate the bands specifically recognized by anti-bFGF antibodies. The positions of the markers for determining mol wt are also depicted.
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1438
LI ET AL.
sensitivity than R30 (Fig. lC), the following experiments were carried out using R30. The elution profile of the immunodetectable bFGF from the heparin-Sepharose affinity column was also examined (Fig. 2). No specific bands could be detected by elution with 1.0 M NaCl, but elution with 2.0 M NaCl gave immunodetectable bFGF from the heparinSepharosecolumn that had been prewashed with 1.0 M NaCl. Again, the high mol wt form of bFGF (24K) was identified as the dominant form in human pituitary adenomas (Fig. 2, lane 5). Next, the molecular forms of bFGF in various pituitary tissues were addressed (Fig. 3). All adenomas (GHproducing, nonsecreting and ACTH-producing adenomas) examined in this experiment were found to contain bFGF with a mol wt of 24K, and one GH-producing tumor showed an additional dense band at the region of 18K (Fig. 3, lane 2). Furthermore, a major form of immunodetectable bFGF in nontumorous anterior pituitary tissues was identified as an
__ *
32.5 27.5 -
JCE & M. 1992 Vol75.No6
18K polypeptide doublet with an additional faint band at 24K region (Fig. 3, lane 6). Mitogenic
activity of human pituitary
adenoma-derived
bFGF
The mitogenic activity in 2 M NaCl extract from human pituitary adenoma was measured(Fig. 4). Two of the extracts used here remarkedly enhanced the DNA synthesis of SC-3 cells in a dose-dependent manner which was in parallel to that elicited by authentic human bFGF. All five adenomas examined showed marked growth-promoting activity (data not shown). The effects of anti-bFGF neutralizing antibody on the growth-promoting activity also were examined using three of the tumor extracts (Fig. 4B). These activities were reduced almost to nonstimulation levels by the addition of
i
0
.OOl
18.5 -
r x1ooo0
.Ol bFGF XlWO
.l
1
10
Xl00
xl0
1 Xl
(nglml)
Dilution FIG. 2. Immunodetectable bFGF in Western blots of heparin-sepharose affinity column eluates of nontumorous or tumorous pituitary extracts. Extract from a nontumorous anterior pituitary gland (lanes 2 and 3) or a GH-producing adenoma (lanes 4 and 5) was diluted to reduce its NaCl concentration to 0.5 M and then applied to a heparinSepharose affinity column. After washing, the materials bound to the column were eluted with 1.0 M NaCl (lanes 2 and 4) and further eluted with 2.0 M NaCl (lanes 3 and 5). These samples as well as authentic bFGF (2 ng; lane 1) were analyzed by the Western blot procedure.
B
123456
Case
FIG. 3. Molecular heterogeneity of bFGF in various pituitary tissues. The mol wt of immunodetectable bFGF in GH-producing (lanes 2 and 4), nonsecreting (lane 3), and ACTH-producing (lane 5) tumors were determined by Western blot analysis. The authentic bFGF (0.5 ng; lane 1) and nontumorous anterior pituitary gland extract (lane 6) were also analyzed.
:I1 no.
6
21
17
bFGF
FIG. 4. Mitogenic activity of FGF-responsive SC-3 cells in pituitary tumor extracts. A, Two pituitary tumor extracts (0 and A) were partially purified by successive passing through heparin-Sepharose, Ampure SA, and Ampure DT columns, and finally eluted with an initial volume of the serum-free medium. After dilution with various volumes of the serum-free medium, their mitogenic activity was measured, as described in Muteriak and Methods. The mitogenic activity of various concentrations of authentic bFGF was also assayed (0). B, Three tumor extracts (cases 6, 21, and 17; see Table 1) were treated as described above. Nondiluted samples were incubated with SC-3 cells in the absence (0) or presence (B) of 50 pg/mL anti-bFGF neutralizing antibody (R & D Systems). SC-3 cells were also stimulated with 0.1 ng/mL bFGF. Their mitogenic activity was measured. All assays were carried out in triplicate. The SES were not depicted because their values were always less than 10%.
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bFGF IN PITUITARY 50 &ml anti-bFGF neutralizing antibody (R & D System). Control rabbit immunoglobulin G failed to inhibit the growth-promoting activity (data not shown). These studies further indicated that human pituitary adenoma can synthesize biologically active bFGF. Northern
blot analysis of FGF receptor
mRNA
and biochemical
analyses in each adenoma
The data on immunodetectable bFGF and FGF receptor mRNA are summarized in Table 1. Clinical profiles also are provided. Immunoreactive bFGF could not be detected in only one nonsecreting adenoma. Four of 10 GH-producing adenomas and 2 of 12 nonsecreting adenomas contained large amounts of 24K bFGF, and 1 ACTH-producing adenoma contained only 24K bFGF. Two nontumorous anterior pituitary glands contained 18K bFGF as a dominant form, with a small quantity of 24K form. We also detected 16K bFGF. This was probably the product of degradation of the higher mol wt forms of bFGF (13). Although qualitative, rather than quantitative, results could be obtained by Western blot analysis, densitometric scanning revealed that some
Caseno.
1 14 13
2
10
11 23
3 16 12 5
FGF Receptor 28s 3.5kb
*
18s
*
p-actin 28s
1439
tumors contained levels of bFGF, similar to those in nontumorous anterior pituitary. Three of 11 adenomas did not possess detectable levels of FGF receptor mRNA. Interestingly, the FGF receptor mRNA level in bFGF-negative nonsecreting adenoma (case 12) was below detectable limits. Discussion
In discussing the biological role of bFGF, the possible presenceof FGF receptor in human pituitary adenomas must not be overlooked. Antibodies to FGF receptor protein, however, were not available, and we had to rely a Northern blot technique in our analysis of FGF receptor mRNA. As shown in Fig. 5, the band hybridizable with chick FGF receptor cDNA was identified at the 3.5 kb region in many, but not all, adenomas. Its size (3.5 kb) was similar to that reported in chick (28), mouse (26), and human (32) cells. Since a relatively large amount of tissue is needed to analyze FGF receptor mRNA, only a limited number of adenomas could be studied. Nonetheless, the results suggest that many adenomas contain FGF receptor. Clinical
ADENOMAS
-
FIG. 5. Northern blot analyses of FGF receptor mRNA in pituitary adenomas. Total RNA from various pituitary adenomas was extracted and electrophoresed. Northern blot analyses were carried out using FGF receptor (upper panel) or P-actin (lower panel) cDNA. These bands were visualized by autoradiography at -80 C for 5 days (FGF receptor) or 18 h (@actin). The positions of 18s and 28s ribosomal RNAs were also depicted. The samples used here were identified by patient case number (see Table 1).
The present studies have clearly demonstrated that human pituitary adenomas contain bFGF. This was confirmed by the following observations. 1) Heparin-binding moleculesin tumor extracts specifically cross-reacted with anti-bFGF antibodies when analyzed by electrophoretic blotting. 2) These immunoreactive molecules were eluted at 1.0-2.0 M NaCl from a heparin-Sepharose affinity column. 3) They were potent mitogens for FGF-responsive SC-3 cells. In addition, the antibodies used here have been reported to be relatively specific for bFGF and not to be reactive to acidic FGF (25); moreover, a certain amount of nonspecific background reaction was observed after immunoblotting, probably because the absolute amount of bFGF in the pituitary tissues was relatively low (33). The major form of bFGF present in the extract from nontumorous anterior pituitary glands was 18K, although we examined only two nontumorous pituitary glands. It is extremely difficult to obtain nontumorous pituitary glands. In addition, postmorten degradation of bFGF molecules(data not shown) prevented us from using autopsy specimens.On the other hand, some human pituitary adenomashave been observed to produce predominantly the high mol wt form (24K) of bFGF. Recently, such high molecular forms (2225K) have been demonstrated in various tissuesor cells (3336). These appear to be the NH2-terminal extended form, derived from a single bFGF mRNA via the use of three CUD initial codons (13, 37). We could identify only two high mol wt forms of bFGF in our human pituitary tissues,probably becausethe mol wt of the two bFGF species(22K and 22.5K) were too similar to obtain sufficient separation. It is still not known whether the NHz-terminus initiates at an AUG codon (18K) or at one of the CUG codons (22K and 24K). Artificial degradation of bFGF from 24K to 18K has been suggestedto occur during the storage of tissuesat -20 C (34). The high mol wt form of guinea pig bFGF can also be converted to an 18K form by trypsin (38). On the other hand, Renko et al. (33) have demonstrated that the control of tryptic activity is not so critical to the distribution of three bFGF forms. While the contribution of protease activities to the molecular heterogeneity of bFGF has not been rigorously eliminated, it should be pointed out that the simultaneously processed adenomas and nontumorous pituitary anterior glands showed clear differences in the mol wt of their bFGF. Our Western blot procedure used here was qualitative, but not quantitative, since the recovery of bFGF was relatively poor (-10%). Also, our data did not demonstrate without doubt that bFGF is present in the adenoma cells as it is in other cell types, such as endothelium, and folliculostellate cells. Even if nontumorous cells are synthesizing bFGF, however, the adenoma cellsmay be exposed to bFGF, sincebFGF is known to adhere to the extracellular matrix (39). In addi-
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TABLE glands
JCE & M - 1992 Vol75.No6
LI ET AL.
1440 1. Characteristics
Case
of immunoreactive
no.
GH-producing adenomas 1 2 3 4 5 6 7 8 9 10 Nonsecreting adenomas 11 12 13 14 15 16 17 18 19 20 21 22 ACTH-producing adenomas 23 Normal anterior pituitary glands 24 25 “The contents blot and classified
bFGF
and FGF
receptor
Tumor (diameter,
Age (yd
Sex
51 38 44 42 30 47 32 49 45 70
M F ii M M M M M M
15 8 15 12 32 24 20 NA 30
58 42 58 67 33 63 68 33 35 39 71 58
M F F M F F F M M M M F
30 18 25 15 40 20 20 NA NA NA NA NA
39
F
13
size mm)
10
mRNA
in human
pituitary
Immunoreactive 16K
18K
adenomas
and normal
bFGF 22K
+++ -
++ ++ +++ ++
pituitary tissues were semiquantified from the densitometric group. ND, Not determined; NA, data were not available.
tion, the FGF receptor is obligatory in eliciting FGF activities. To our knowledge, this is the first report to demonstrate the presence of FGF receptor mRNA in human pituitary adenoma. Interestingly, our bFGF-negative tumors did not contain detectable levels of FGF receptor mRNA. In another study we demonstrated that bFGF can up-regulate FGF receptor mRNA in SC-3 cells (26). These findings tempt us to speculate that the lack of bFGF synthesis arrests FGF receptor mRNA expression. The biological role of bFGF in the growth and function of normal or transformed pituitary cells has been mainly examined by NH2-terminal deleted forms (16K and 18K) of bFGF in the culture medium. However, a recent finding has suggested the existence of different signal pathways for bFGF, depending on whether bFGF is produced by or provided to the cells (40). Moreover, the NH2-terminus present in a 24K form of bFGF has been proven to contain the nuclear localization signal (14, 33). The subcellular and extracellular localization of bFGF in the development and progression of pituitary adenoma should be examined in future studies. Acknowledgments The authors acknowledge Prof. T. Kishimoto for his encouragement for the present study. We also thank our clinical staff for taking care of the patients and Mr. D. Elick for his editorial help.
pituitary
FGF receptor mRNA
24K
+++ ++ ++
39 38
of each mol wt form of bFGF in human +, ++, and +++ in the bFGF-positive
anterior
+++ ++
+++ +++ + +++ ++ ++
ND ++ ND ND ND ND -
++ +++ ++ +++ ++ -
+++ ++ ++ ND ++ ND ND ND ND ND ND
+++
++
+ ++
ND ND scanning
data on Western
References 1. Gospodarowicz D. 1974 Localization of a fibroblast growth factor and its effect alone and with hydrocortisone on 3T3 cell growth. Nature. 249:123. 2. Esch F, Baird A, Ling N, et al. 1985 Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Proc Nat1 Acad Sci USA. 82:6507-6511, 3. Kasselberg AG, Orth DN, Gray ME, Stablman MT. 1985 Immunocytochemical localization of human epidermal growth factor/ urogastrone in several human tissues. J Histochem Cytochem. 33:315-322. 4. Binoux M., Hossenlopp P, Lassarre C, Hardouin N. 1981 Production of insulin-like growth factors and their carrier by rat pituitary gland and brain explants in culture. FEBS Lett. 124:178-184. 5. Hasselbacher GK, Schwab ME, Psai A, Humbel RE. 1985 Insulinlike growth factor II (IGF-II) in human brain: region distribution of IGF-II and higher molecular mass forms. Proc Nat1 Acad Sci USA. 82:2153-2157. 6. Kobrin MS, Asa SL, Samsoondar J, Kudlow JE. 1987 a-Transforming growth factor in the bovine anterior pituitary gland: secretion by dispersed cells and immunohistochemical localization. Endocrinology. 121:1412-1416. 7. Lahtinen T, Soinila S, Lakshmanan J. 1989 Biological demonstration of nerve growth factor in the rat pituitary gland. Neuroscience. 30:165-170. 8. Gospodarowicz D. 1975 Purification of a fibroblast growth factor from bovine pituitary. J Biol Chem. 250:2515-2520. 9. Schonbrunn B, Krasnoff M, Westendonf JM, Tashjiian AH. 1980 Epidermal growth factor and thyrotropin-releasing hormone act similarly on clonal pituitary cell strain. J Cell Biol. 85:786-797.
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bFGF
IN PITUITARY
10. Baird A, Mormede P, Ying S-Y, et al. 1985 A nonmitogenic pituitary function of fibroblast growth factor: regulation of thyrotropin and prolactin secretion. Proc Nat1 Acad Sci USA. 82:5545-5549. 11. Mormede P, Baird A. 1988 Estrogen, cyclic adenosine 3’,5’-monophosphate, and phorbol esters modulate the prolactin response of GH, cells to basic fibroblast growth factor. Endocrinology. 12212265-2271. 12. Abraham JA, Whang JL, Tumolo A, Mergia A, et al. 1986 Human basic fibroblast growth factor: nucleotide sequence and genomic organization. EMBO J. 5:2523-2528. 13. Florkiewicz RZ, Sommer A. 1989 Human basic fibroblast growth factor gene encodes four polypeptides: three initiate translation from non-AUG codons. Proc Nat1 Acad Sci USA. 86:3978-3981. 14. Quart0 N, Finger FP, Rifkin DB. 1991 The NHz-terminal extension of high molecular weight basic fibroblast growth factor is a nuclear targeting signal. J Cell Physiol. 147:311-318. 15. Imamura T, Engleka K, Zhan X, et al. 1990 Recovery of mitogenic activity of a growth factor mutant with a nuclear translocation sequence. Science. 249:1567-1570. 16. Herman V, Fagin J, Gonsky R, Kovacs K, Melmed S. 1990 Clonal origin of pituitary adenomas. J Clin Endocrinol Metab. 71:14271433. 17. Alexander JM, Biller BM, Bikkal H, Zervas NT, Arnold A, Klibanski A. 1990 Clinically nonfunctioning pituitary tumors are monoclonal in origin. J Clin Invest. 86:336-340. 18. Cross M, Dexter TM. 1991 Growth factors in development, transformation, and tumorigenesis. Cell. 64:271-280. 19. Kaspar S, Friesen HG. 1986 Human pituitary tissue secretes a potent growth factor for chondrocyte proliferation. J Clin Endocrinol Metab. 62:70-76. J, Ham J, Bevan JS, ten Horn CD, Scanlon MF. 1991 20. Webster Preliminary characterization of growth factors secreted by human pituitary tumors. J Clin Endocrinol Metab. 72:687-692. JJ, Prysor-Jones RA, Jenkins JS. 1990 Basic fibroblast 21. Silverlight growth factor in human pituitary tumors. Clin Endocrinol (Oxf). 321669-676. 22. Mori H, Mori S, Saitoh Y, et al. 1985 Effect of bromocriptine on prolactin-secreting pituitary adenoma. Cancer. 56:230-238. 23. Koga M, Nakao H, Arao M, et al. 1987 Demonstration of specific dopamine receptors on human pituitary adenomas. Acta Endocrinol (Copenh). 114:595-602. 24. Laemmli UK. 1970 Cleavage of structural proteins during the assembIy of the head of bacteriophage T+ Nature. 227:680-685. 25. Klagsbrun M, Sasse J, Sullivan R, Smith JA. 1986 Human tumor cells synthesize an endothelial cell growth factor that is structurally related to basic fibroblast erowth factor. Proc Nat1 Acad Sci USA. 83:2448-2452. 26. Saito H, Kasayama S, Kouhara H, Matsumoto K, Sato B. 1991 Upregulation of fibroblast growth factor (FGF) receptor mRNA levels by basic FGF or testosterone in androgen-sensitive mouse
ADENOMAS
27.
28.
29.
30
31
32
33
34
35
36.
37.
38.
39. 40.
1441
mammary tumor cells. Biochem Biophys Res Commun. 174:136141. Chirgwin J, Pryzbyla A, McDonald R, Rutter W. 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 18:5294-5299. Lee PL, Johnson DE, Cousens LS, Fried VA, Williams LT. 1989 Purification and complementary DNA cloning of a receptor for basic fibroblast growth factor. Science. 245:57-60.Noeuchi S, Nishizawa Y, Nakamura N, et al. 1987 Growthstimulating effect of pharmacological doses of estrogen on androgen-dependent Shionogi carcinoma 115 in vivo but not in cell culture. Cancer Res. 47:263-268. Nakamura N, Yamanishi H, Lu J, et al. 1989 Growth-stimulatory effects of androgen and high concentration of glucocorticoid or fibroblast growth factors on a cloned cell line from Shionogi carcinoma 115 cells in a serum-free medium. J Steroid Biochem. 33:1318. Kasayama S, Sumitani S, Matsumoto K, Sato B. 1991 Heparin inhibits autocrine stimulation but not fibroblast growth factor stimulation of cell proliferation of androgen-responsive Shionogi carcinoma 115. J Cell I’hysiol. 148:260-266. Houssaint E, Blanquet PR, Champion-Aranaud P, et al. 1990 Related fibroblast growth factor receptor genes exist in the human eenome Proc Nat1 Acad Sci USA. 87:8180-8184. Renko M, Quart0 N, Morimoto T, Rifkin DB. 1990 Nuclear and cytoplasmic localization of different basic fibroblast growth factor species. J Cell Physiol. 144:108-114. Presta M, Statuto M, Rusnati M, Dell’Era P, Ragnotti G. 1989 Characterization of a Mr 25,000 basic fibroblast growth factor form in adult, regenerating, and fetal rat liver. Biochem Biophys Res Commun. 164:1182-1189. Sommer A, Moscatelli D, Rifkin DB. 1989 An amino-terminally extended and post-translationally modified form of a 25 Kd basic fibroblast growth factor. Biochem Biophys Res Commun. 160:12671274. Maier JAM, Rusnati M, Ragnotti G, Presta M. 1990 Characterization of a Mr 20,000 basic fibroblast growth factor-like protein secreted by normal and transformed fetal bovine aortic endothelial cells, Exp Cell Res. 186:354-361. Prats H, Kaghad H, Prats AC, et al. 1989 High molecular mass forms of ba& fibroblast growth factor are initiated by alternative CUG codons. Proc Nat1 Acad Sci USA. 86:1836-1840. Moscatelli D, Joseph-Silverstein J, Manejias R, Rifkin DB. 1987 Mr 25,000 heparin:binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor. Proc Nat1 Acad Sci USAu84:5778-5782. Rifkin DB, Moscatelli D. 1989 Recent developments in the cell biology of basic fibroblast growth factor. J Cell Biol. 109:1-6. Burgess WH, Maciag T. 1989 The heparin-binding (fibroblast) family of proteins. Annu Rev Biochem. 58:575-606.
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