Clinical Endocrinology (1990), 32,669-676
BASIC FIBROBLAST GROWTH FACTOR IN HUMAN PITUITARY TUMOURS J. J. SILVERLIGHT, R. A . PRYSOR-JONES
AND
J. S. J E N K I N S
Division of Biochemical Medicine, St. George’s Hospital Medical School, Cranmer Terrace, London S WI 7 ORE, UK (Received 15 September 1989: returned for revision 13 November 1989;finally revised 24 November 1989; accepted 14 December 1989)
SUMMARY
Immunoreactive (ir) basic fibroblast growth factor (FGF) has been measured in normal human pituitary glands and in 41 pituitary tumours of various types using a radioimmunoassay. Normal pituitary glands contained ir basic F G F ranging from 29.4 to 355 fmol/mg wet weight whereas 87.5% of pituitary tumours contained amounts of the growth factor which were less than normal. There was no relationship between the type of tumour and the FGF content. Normal and tumorous pituitary FGF had an affinity for heparin which was similar to that of basic FGF. Gel column chromatography revealed that the pituitary ir FGF was present mainly as high molecular weight forms, with only a small proportion as the basic 146 amino-acid peptide. Because of our previous observation that basic FGF inhibits the growth of human pituitary tumour cells, it is suggested that the reduction in basic F G F of many pituitary tumours may favour the stimulation of pituitary growth. Fibroblast growth factor (FGF) was first reported to be present in high concentrations in the bovine pituitary gland by Gospodarowicz (1974) and was subsequently characterized as basic FGF, which is a straight-chain peptide composed of 146 amino-acids (Esch et al., 1985). Further studies have revealed that basic FGF also exists as N-terminal-truncated or extended forms in some tissues, including the pituitary (Ueno et al., 1986; Bertolini & Hearn, 1987). Another form of F G F having a 55% homology in structure with the basic form and designated acidic FGF was isolated from brain tissue (Giminez-Galego et al., 1985), although its presence in pituitary tissue is controversial (Gambarini & Armelin, 1982; Gospodarowicz et al., 1987). A common characteristic of the various forms of FGF is their strong affinity for heparin, and this property is used for their purification. Fibroblast growth factors have been identified in a large variety of tissues. They have a wide spectrum of biological activity and are mitogenic not only in fibroblasts but in many mesodermal and neuroectodermal-derived cell types (Gospodarowicz et al., 1987; Rifkin & Moscatelli, 1989). However, in spite of its isolation from normal bovine pituitary Correspondence: Professor J. S . Jenkins, Division of Biochemical Medicine, St George’s Hospital Medical School, Cranmer Terrace, London SW17 ORE, UK.
669
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J. J. Silverlight et al.
glands several years ago there have been few studies on the effects of basic FGF on pituitary growth. As part of an investigation into its role in the development of pituitary tumours we have used a radioimmunoassay for basic FGF to determine its concentration in different types of human pituitary adenomas. We have also investigated the molecular form of FGF within pituitary tissue. MATERIALS A N D METHODS Human pituitary tumour samples were obtained at surgery and immediately frozen. The nature of the tumour was routinely determined by the circulating hormone concentration and by immunocytochemical examination. Normal human pituitary glands were obtained post-mortem from patients without pituitary disease 6-24 h after death. Recombinant 1-146 basic FGF was obtained from Amersham International plc, Amersham, Bucks., UK. Antibody to FGF 1-24 raised in rabbits was kindly given by Dr. A. Baird of the Salk Institute, USA. This antibody also reacts with the extended 155 and 157 amino-acid forms of basic FGF, but not with the truncated molecule (Baird, personal communication). Radioimmunoassay of basic FGF
The method was modified from that of Baird et al. (1985a). Recombinant basic FGF 1146 was labelled with '251by the iodogen method (Salacinski et al., 1979). Radioactively labelled FGF was purified by loading on a heparin-Sepharose column, washing free of iodine with 0 . 6 NaCl ~ and eluting with 2 ml2M NaCI. The labelled peptide was stable for 2-3 weeks at - 70°C. Incubations of serial dilutions of FGF in 10 mM phosphate buffer containing 0.1 % (w/v) gelatin were carried out at pH 7.2, with antibody in a final dilution of 1 : 45 000 and approximately 10 000 c.p.m. radioactively labelled FGF. The tubes were incubated at 4°C for 24 h and the bound and free antigen were separated using a second antibody (SacCell anti-rabbit serum, IDS, Washington, Tyne and Wear, UK). Cross-reactivity with acidic FGF was < 0.1%. The sensitivity of the assay was 8 fmol/ tube and the coefficients of variation were 4.9% for intra-assays and 14.0% for interassays in the range 50-1 50 fmol. The recovery of standard FGF added to tissue homogenate in this range was 82-89%. Results were not corrected for recovery. Preparation of tissue extracts
Normal pituitary or tumour tissue samples were weighed and homogenized in 10 volumes of ammonium sulphate (0.15 ~at)pH 7.0 at 4°C. Homogenates were centrifuged at 30 000 g for 30 min at 4°C and aliquots of the supernatant were taken for assay. Heparin afinity chromatography
Heparin-agarose (Sigma Chemicals, Poole, Dorset) was washed with 10 volumes of 0 . 0 5 ~ phosphate buffer, pH 7.5 and 0.2 ml was mixed with 1 ml supernatant from pituitary extracts for 30 min at 4°C. This was transferred to a mini-column, 0.7 cm diameter, and ~ in 1 ml steps, increasing by 0 . 1 ~Over . 95% of ir basic FGF eluted with 0 . 1 to ~ 3 . 0 NaCl was shown to be bound to the heparin-agarose before elution.
Fibroblast growth factor in pituitary tumours 100 80
-3
60
-
0
m 40 -
:I1,
20 O I- 10
671
:“.h. I00
1000
I
I 1000
I00 ir bFGF (frnolltube)
Fig. I . 0 , Standard curve for ir basic FGF; showing parallelism.
.,
serial dilutions of extract of pituitary tumour
Gel column chromatography Aliquots of the supernatant were loaded on Sephacryl S-300 (Pharmacia, Uppsala, Sweden) 60 x 0.9 cm columns and eluted with sodium citrate buffer ( 0 4 2 M ) at pH 5.0. One ml fractions of eluate were collected for assay.
I
.. .
.
150-
A
.
i I +
a
250
3
LL P L .-
100 t
50
-
.
.....*. :..
..*...*!Ito:.:
., :
Fig. 2. Ir basic FGF in a, normal and b-e, tumorous human pituitaries. b, Non-functional; c. GH; d. PRL; e, ACTH.
672
J . J . Silverlight et al. RESULTS Measurement of basic FGF in pituitary tissue
A standard curve for ir basic FGF is shown in Fig. 1 which indicates that there was complete parallelism with dilutions of pituitary extract. Normal pituitary
In 15 normal pituitary glands ir FGF ranged from 29.4 to 355 fmol/mg wet weight (median 150, Fig. 2). There was no relationship with age, sex, or duration post-mortem. Pituitary tumours
Forty-one tumours were studied from patients whose ages ranged from 30 to 73 years (median 44). There were 23 non-functioning tumours including one from a patient with
01 1.0
1.5
2.0
2.5
3-0
NaCl (rnol/l)
Fig. 3. Heparin-affinity chromatography of a, ir basic FGF; b, normal pituitary; c, tumorous pituitary extract.
Fibroblast growth factor in pituitary tumours
.a
“I
I
2
3
4
I
I I
I
673
4
I
(b’
20
25
30 35 40 Fraction number
45
Fig. 4. Gel-filtration column chromatography of a, basic FGF; b, pituitary extract on Sephacryl S-300 columns. Molecular weight markers (kDa): 1, blue dextran (2000); 2, globulin (150); 3, bovine serum albumin (67); 4, cytochrome c (12.4).
pituitary apoplexy, 11 growth hormone secreting (plasma growth hormone range 13.1650 mU/1), five prolactin-secreting (plasma prolactin range 8000-90 000 mU/l) and two ACTH-secreting tumours (plasma ACTH 196 and 140 mg/l). Concentrations of ir basic FGF within the tumours are shown in Fig. 2. The values ranged from an immeasurably small amount, which was found in the tumour which had undergone infarction and haemorrhage, to 308 fmol/mg. However, in 36 tumours (87.5%) the values were less than normal, and in five, values were within the normal range. There was no relationship between the ir basic FGF concentration and the hormonal status of the tumour, or the age or sex of the patients. The two highest values were present in tumours with circulating growth hormone of 12.3 mU/1 and prolactin of 8000 mU/1 respectively but the other tumours in these categories included some with much greater circulating hormone levels and low amounts of ir basic FGF. Heparin-afinity chromatography of pituitary FGF By the use of heparin-agarose column chromatography it was shown that ir basic FGF in both normal and tumorous human pituitary extracts eluted maximally with 1-6-1.8~ NaCl and there was a smaller peak at 2 . 5 ~ NaCI. This profile was similar to that of authentic 1-146 basic FGF (Fig. 3). Molecular size of ir basic FGF in human pituitary tissue
Column chromatography profiles of extracts from a normal pituitary gland compared with ir basic FGF are shown in Fig. 4. Most of the pituitary ir basic FGF was present in a
614
J . J . Silverlight et al.
broad region corresponding to a molecular weight ranging from about 70-1 50 kDa and only a small amount at the position of authentic 146 amino-acid FGF, indicating that much of the ir basic FGF was present in high molecular weight forms. DISCUSSION These results show that ir basic FGF is present in normal human pituitary tissue in readily measurable concentrations. It has previously been shown that basic FGF is very stable at neutral pH in body fluids (Baird et al., 1986) so that the use of post-mortem material of 624 h duration appears to be justified. In human pituitary tumours there was a wide range of concentrations of ir basic FGF but the important finding was that in 87.5% the values were lower than those in normal tissue. Although it has been reported that FGF can increase the release of prolactin and decrease that of growth hormone from a rat pituitary tumour cell line (Schonbrunn et al., 1980) and can increase the prolactin response to TRH of normal rat pituitaries (Baird et al., 1985b), we could find no relationship between ir basic FGF content and the hormonal characteristics of the human tumours. The two tumours with ir basic FGF values which were much higher than the others were growth hormone or prolactin-secreting but there were no distinguishing features, either clinically or histologically, about these particular tumours. One of the characteristics of FGF is its strong affinity for heparin and in this respect ir FGF in pituitary extracts behaved in an identical manner to that of standard basic FGF, eluting maximally at 1.6-1.8~NaCl. In addition there was a smaller amount of material ~ This ir FGF has also been with a stronger affinity for heparin eluting at about 2 . 5 NaC1. reported by Baird et al. (1986) and is presumed to be another microheterogeneous form. The molecular form of ir basic FGF within the pituitary was found to be greatly different from that of authentic 1-146 FGF which has a molecular weight of approximately 16 kDa. Most of the ir basic FGF was present as a large molecular form and in the region of 70-150 kDa. Mormede et al. (1985) have reported similar high molecular weight forms of basic FGF in a variety of rat tissues including the pituitary gland of this species and they identified no FGF of 16 kDa molecular weight. The nature of the high molecular weight forms is not known, but it is significant that the glycosaminoglycan heparin to which FGF has a strong affinity is closely related to heparin sulphate (Kraemer, 1971) which is a component of the extracellular matrix of tissues. Recent work has shown that in vivo basic FGF binds strongly to glycosaminoglycans in the extracellular matrix of bovine endothelial cells (Baird & Ling, 1987) or the basement membrane-like matrix of a mouse sarcoma (Vigny et al., 1988). This extracellular binding can be prevented by degradation of the heparin sulphate chains with the enzyme heparitinase. It seems possible, therefore, that at least some of the high molecular weight forms of ir basic FGF found in pituitary tissue may represent binding to the glycosaminoglycans of the extracellular matrix. In view of the finding that FGF appears to be present extracellularly in many tissues, its site of origin within the pituitary requires consideration. Little or no basic FGF has been found in rat pituitary cell lines (Baird et al., 1986) and we have been unable to detect basic FGF either within cells or in the medium from cultures of a human prolactin-secreting tumour cell line (unpublished). Recently, Ferrara et al. (1987) have produced evidence that a separate population of pituitary cells, designated folliculo-stellate cells, are a major
Fibroblast growth factor in pituitary tumours
675
source of basic FGF within the normal pituitary. Our finding of reduced amounts of ir basic F G F in tumorous pituitaries would be in agreement with this view since folliculostellate cells are not a predominant feature of adenomatous tissue. Whatever the site of origin, it is suggested by Baird and Ling (1987) that after its expression basic FGF would become associated with the glycosaminoglycans of the extracellular matrix or basement membrane to act as a reservoir for the growth factor. From this complex it would then be released by local enzyme action to act on specific cell surface F G F receptors to induce biological activity. The function of basic F G F in pituitary tumours, or indeed in normal pituitary glands, is not clear. However, recent work by us has shown that in contrast with its mitogenic activity on fibroblasts, basic 1-146 FGF inhibits the DNA synthesis of human pituitary tumour cells, whereas a different heparin-binding factor derived from pituitary glands is stimulatory to these same cells (Prysor-Jones et al., 1989). Schweigerer et al. (1987) have reported that basic F G F inhibits the growth of a human Ewing’s tumour cell line in culture but is stimulatory to some other tumours. It is clear, therefore, that the effect of basic F G F is complex since it can be stimulatory or inhibitory towards cell growth depending upon the particular cell type. The reason for this bifunctional effect is not known. We suggest, however, that in the case of pituitary tumours the reduction in ir basic F G F which we have demonstrated to be present in many instances would lead to less restraint of cell growth and the balance would then be tipped in favour of stimulatory factors. ACKNOWLEDGEMENTS
We are grateful to the Medical Research Council for financial support and to Dr A. Baird for antisera. REFERENCES BAIRD, A,, BOHLEN. P.,LING,N. & GUILLEMIN, R. (1985a) Radioimmunoassay for fibroblast growth factor (FGF): release by the bovine anterior pituitary in vitro. Regulatory Peptides, 10, 309-3 17. BAIRD,A., ESCH,F., MORMEDE, P., UENO,N., LING,N., BOHLEN,P., YING,S.Y., WEHRENBERG, W.B. & GUILLEMIN, R. (1986) Molecular characterization of fibroblast growth factor: distribution and biological activities in various tissues. Recent Progress in Hormone Research, 42, 143-205. BAIRD,A. & LING,N . (1987) Fibroblast growth factors are present in the extracellular matrix produced by endothelial cells in oitro. Biochemical and Biophysical Research Communications, 142,428-634. BAIRD, A., MORMEDE, P., YING,S.Y., WEHRENBERG, W.B., UENO,N., LING,N. &GUILLEMIN, R. (1985b)A nonmitogenic pituitary function of fibroblast growth factor: regulation of thyrotropin and prolactin secretion. Proceedings of National Academy of Sciences ofthe USA, 82, 554555549, BERTOLINI, J. & HEARN,M.T.W. (1987) Isolation, characterisation, and tissue localisation of an N-terminaltruncated variant of fibroblast growth factor. Molecular and Cellular Endocrinology, 51, 187-199. ESCH,F.,BAIRD, A., LING,N., UENO,N.,HILL,F.. DENEROY, L., KLEPPER, R., GOSFQDAROWICZ, D., BOHLEN, P. & GUILLEMIN, R. (1985) Primary structure of bovine pituitary fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Proceedings of National Academy of Sciences of the USA, 85,6507-651 1. FERRARA, N . , SCHWEIGERER, L.S., NEUFELD, G., MITCHELL, R. & GOSPODAROWITZ, D. (1987) Pituitary follicular cells produce fibroblast growth factor. Proceedings of National Academy ofsciences of the USA, 84, 5713-5711. GAMBARINI, A.G. & ARMELIN, H.S. (1982) Purification and partial characterization of an acidic fibroblast growth factor from bovine pituitary. Journal of Biological Chemistry, 257, 9692-9697.
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GIMEZ-GALLEGO, G., RODKEY, J., BENNETT, C., RIOS-CANDELORE, M., DISALVO, J. &THOMAS, K. (1985) Brain derived acidic fibroblast growth factor: complete amino acid sequence and homologies. Science, 230,13851388. GOSPODAROWICZ, D. (1974) Purification of a fibroblast growth factor from bovine pituitary. Journal of Biological Chemistry, 250,251 5-2520. GOSPODAROWICZ, D., FERRARA, N., SCHWEIGERER, L. & NEUFELD, G. (1987) Structural characterization and biological functions of fibroblast growth factor. Endocrine Reviews, 8,95-113. KRAEMER, P.M. (1971) Heparin-sulphates of cultured cells. Biochemistry, 10, 1437-1443. MORMEDE, P., BAIRD,A. & PIGEON, P. (1985) Immunoreactive fibroblast growth factor (FGF) in rat tissues: molecular weight forms and the effects of hypophysectomy. Biochemical and Biophysicar Research Communications, 128, 1108-1 113. PRYSOR-JONES R.A., SILVERLIGHT, J.J. & JENKINS, J.S. (1989) Oestradiol, vasoactive intestinal peptide and fibroblast growth factor in the growth of human pituitary tumour cells in uifro.Journal of Endocrinology, 120,171-177. RIFKIN,D.B. & MOSCATELI, D. (1989) Recent developments in the cell biology of basic fibroblast growth factor. Journal of Cell Biology, 109, 1-6. SALACINSKI, P., HOPE,J., MCLEAN,C., CLEMENT-JONES, V., SYKES,J., PRICE,J. & LOWRY,P.J. (1979) A new simple method which allows theoretical incorporation of radioiodine into proteins and peptides without damage. Journal of Endocrinology, 81, 131P. SCHONBRUNN, A,, KRASNOFF, M., WESTENWRF, J.M. & TASHJIAN, A.H. (1980) Epidermal growth factor and thyrotropin releasing hormone act similarly on a clonal pituitary cell strain. Journal of Cell Biology, 85, 786-797. SCHWEIGERER, L., NELIFELD,G. & GOSPODAROWCZ, D. (1987) Basic fibroblast growth factor as a growth inhibitor for cultured human tumour cells. Journal of Clinical Invesfigafion,80, 1516-1520. UENO,N., BAIRD,A,, ESCH,F., LING,N. & GUILLEMIN, R. (1986) Isolation of an aminoterminal extended form of basic fibroblast growth factor. Biochemical and Biophysical Research Communications, 138,580-588. VIGNY,M., OLLIER-HARTMANN, M.P., LAVIGNE, M., FAYEIN,N., JEANNY, J.C., LAURENT,M. & COURTOIS, Y. (1988) Specific binding of fibroblast growth factor to purified heparin-sulfate proteoglycan of the EHS tumor. Journal of Cellular Physiology, 137,32 1-328.
BASIC FIBROBLAST GROWTH FACTOR IN HUMAN PITUITARY TUMOURS J. J. SILVERLIGHT, R. A . PRYSOR-JONES
AND
J. S. J E N K I N S
Division of Biochemical Medicine, St. George’s Hospital Medical School, Cranmer Terrace, London S WI 7 ORE, UK (Received 15 September 1989: returned for revision 13 November 1989;finally revised 24 November 1989; accepted 14 December 1989)
SUMMARY
Immunoreactive (ir) basic fibroblast growth factor (FGF) has been measured in normal human pituitary glands and in 41 pituitary tumours of various types using a radioimmunoassay. Normal pituitary glands contained ir basic F G F ranging from 29.4 to 355 fmol/mg wet weight whereas 87.5% of pituitary tumours contained amounts of the growth factor which were less than normal. There was no relationship between the type of tumour and the FGF content. Normal and tumorous pituitary FGF had an affinity for heparin which was similar to that of basic FGF. Gel column chromatography revealed that the pituitary ir FGF was present mainly as high molecular weight forms, with only a small proportion as the basic 146 amino-acid peptide. Because of our previous observation that basic FGF inhibits the growth of human pituitary tumour cells, it is suggested that the reduction in basic F G F of many pituitary tumours may favour the stimulation of pituitary growth. Fibroblast growth factor (FGF) was first reported to be present in high concentrations in the bovine pituitary gland by Gospodarowicz (1974) and was subsequently characterized as basic FGF, which is a straight-chain peptide composed of 146 amino-acids (Esch et al., 1985). Further studies have revealed that basic FGF also exists as N-terminal-truncated or extended forms in some tissues, including the pituitary (Ueno et al., 1986; Bertolini & Hearn, 1987). Another form of F G F having a 55% homology in structure with the basic form and designated acidic FGF was isolated from brain tissue (Giminez-Galego et al., 1985), although its presence in pituitary tissue is controversial (Gambarini & Armelin, 1982; Gospodarowicz et al., 1987). A common characteristic of the various forms of FGF is their strong affinity for heparin, and this property is used for their purification. Fibroblast growth factors have been identified in a large variety of tissues. They have a wide spectrum of biological activity and are mitogenic not only in fibroblasts but in many mesodermal and neuroectodermal-derived cell types (Gospodarowicz et al., 1987; Rifkin & Moscatelli, 1989). However, in spite of its isolation from normal bovine pituitary Correspondence: Professor J. S . Jenkins, Division of Biochemical Medicine, St George’s Hospital Medical School, Cranmer Terrace, London SW17 ORE, UK.
669
610
J. J. Silverlight et al.
glands several years ago there have been few studies on the effects of basic FGF on pituitary growth. As part of an investigation into its role in the development of pituitary tumours we have used a radioimmunoassay for basic FGF to determine its concentration in different types of human pituitary adenomas. We have also investigated the molecular form of FGF within pituitary tissue. MATERIALS A N D METHODS Human pituitary tumour samples were obtained at surgery and immediately frozen. The nature of the tumour was routinely determined by the circulating hormone concentration and by immunocytochemical examination. Normal human pituitary glands were obtained post-mortem from patients without pituitary disease 6-24 h after death. Recombinant 1-146 basic FGF was obtained from Amersham International plc, Amersham, Bucks., UK. Antibody to FGF 1-24 raised in rabbits was kindly given by Dr. A. Baird of the Salk Institute, USA. This antibody also reacts with the extended 155 and 157 amino-acid forms of basic FGF, but not with the truncated molecule (Baird, personal communication). Radioimmunoassay of basic FGF
The method was modified from that of Baird et al. (1985a). Recombinant basic FGF 1146 was labelled with '251by the iodogen method (Salacinski et al., 1979). Radioactively labelled FGF was purified by loading on a heparin-Sepharose column, washing free of iodine with 0 . 6 NaCl ~ and eluting with 2 ml2M NaCI. The labelled peptide was stable for 2-3 weeks at - 70°C. Incubations of serial dilutions of FGF in 10 mM phosphate buffer containing 0.1 % (w/v) gelatin were carried out at pH 7.2, with antibody in a final dilution of 1 : 45 000 and approximately 10 000 c.p.m. radioactively labelled FGF. The tubes were incubated at 4°C for 24 h and the bound and free antigen were separated using a second antibody (SacCell anti-rabbit serum, IDS, Washington, Tyne and Wear, UK). Cross-reactivity with acidic FGF was < 0.1%. The sensitivity of the assay was 8 fmol/ tube and the coefficients of variation were 4.9% for intra-assays and 14.0% for interassays in the range 50-1 50 fmol. The recovery of standard FGF added to tissue homogenate in this range was 82-89%. Results were not corrected for recovery. Preparation of tissue extracts
Normal pituitary or tumour tissue samples were weighed and homogenized in 10 volumes of ammonium sulphate (0.15 ~at)pH 7.0 at 4°C. Homogenates were centrifuged at 30 000 g for 30 min at 4°C and aliquots of the supernatant were taken for assay. Heparin afinity chromatography
Heparin-agarose (Sigma Chemicals, Poole, Dorset) was washed with 10 volumes of 0 . 0 5 ~ phosphate buffer, pH 7.5 and 0.2 ml was mixed with 1 ml supernatant from pituitary extracts for 30 min at 4°C. This was transferred to a mini-column, 0.7 cm diameter, and ~ in 1 ml steps, increasing by 0 . 1 ~Over . 95% of ir basic FGF eluted with 0 . 1 to ~ 3 . 0 NaCl was shown to be bound to the heparin-agarose before elution.
Fibroblast growth factor in pituitary tumours 100 80
-3
60
-
0
m 40 -
:I1,
20 O I- 10
671
:“.h. I00
1000
I
I 1000
I00 ir bFGF (frnolltube)
Fig. I . 0 , Standard curve for ir basic FGF; showing parallelism.
.,
serial dilutions of extract of pituitary tumour
Gel column chromatography Aliquots of the supernatant were loaded on Sephacryl S-300 (Pharmacia, Uppsala, Sweden) 60 x 0.9 cm columns and eluted with sodium citrate buffer ( 0 4 2 M ) at pH 5.0. One ml fractions of eluate were collected for assay.
I
.. .
.
150-
A
.
i I +
a
250
3
LL P L .-
100 t
50
-
.
.....*. :..
..*...*!Ito:.:
., :
Fig. 2. Ir basic FGF in a, normal and b-e, tumorous human pituitaries. b, Non-functional; c. GH; d. PRL; e, ACTH.
672
J . J . Silverlight et al. RESULTS Measurement of basic FGF in pituitary tissue
A standard curve for ir basic FGF is shown in Fig. 1 which indicates that there was complete parallelism with dilutions of pituitary extract. Normal pituitary
In 15 normal pituitary glands ir FGF ranged from 29.4 to 355 fmol/mg wet weight (median 150, Fig. 2). There was no relationship with age, sex, or duration post-mortem. Pituitary tumours
Forty-one tumours were studied from patients whose ages ranged from 30 to 73 years (median 44). There were 23 non-functioning tumours including one from a patient with
01 1.0
1.5
2.0
2.5
3-0
NaCl (rnol/l)
Fig. 3. Heparin-affinity chromatography of a, ir basic FGF; b, normal pituitary; c, tumorous pituitary extract.
Fibroblast growth factor in pituitary tumours
.a
“I
I
2
3
4
I
I I
I
673
4
I
(b’
20
25
30 35 40 Fraction number
45
Fig. 4. Gel-filtration column chromatography of a, basic FGF; b, pituitary extract on Sephacryl S-300 columns. Molecular weight markers (kDa): 1, blue dextran (2000); 2, globulin (150); 3, bovine serum albumin (67); 4, cytochrome c (12.4).
pituitary apoplexy, 11 growth hormone secreting (plasma growth hormone range 13.1650 mU/1), five prolactin-secreting (plasma prolactin range 8000-90 000 mU/l) and two ACTH-secreting tumours (plasma ACTH 196 and 140 mg/l). Concentrations of ir basic FGF within the tumours are shown in Fig. 2. The values ranged from an immeasurably small amount, which was found in the tumour which had undergone infarction and haemorrhage, to 308 fmol/mg. However, in 36 tumours (87.5%) the values were less than normal, and in five, values were within the normal range. There was no relationship between the ir basic FGF concentration and the hormonal status of the tumour, or the age or sex of the patients. The two highest values were present in tumours with circulating growth hormone of 12.3 mU/1 and prolactin of 8000 mU/1 respectively but the other tumours in these categories included some with much greater circulating hormone levels and low amounts of ir basic FGF. Heparin-afinity chromatography of pituitary FGF By the use of heparin-agarose column chromatography it was shown that ir basic FGF in both normal and tumorous human pituitary extracts eluted maximally with 1-6-1.8~ NaCl and there was a smaller peak at 2 . 5 ~ NaCI. This profile was similar to that of authentic 1-146 basic FGF (Fig. 3). Molecular size of ir basic FGF in human pituitary tissue
Column chromatography profiles of extracts from a normal pituitary gland compared with ir basic FGF are shown in Fig. 4. Most of the pituitary ir basic FGF was present in a
614
J . J . Silverlight et al.
broad region corresponding to a molecular weight ranging from about 70-1 50 kDa and only a small amount at the position of authentic 146 amino-acid FGF, indicating that much of the ir basic FGF was present in high molecular weight forms. DISCUSSION These results show that ir basic FGF is present in normal human pituitary tissue in readily measurable concentrations. It has previously been shown that basic FGF is very stable at neutral pH in body fluids (Baird et al., 1986) so that the use of post-mortem material of 624 h duration appears to be justified. In human pituitary tumours there was a wide range of concentrations of ir basic FGF but the important finding was that in 87.5% the values were lower than those in normal tissue. Although it has been reported that FGF can increase the release of prolactin and decrease that of growth hormone from a rat pituitary tumour cell line (Schonbrunn et al., 1980) and can increase the prolactin response to TRH of normal rat pituitaries (Baird et al., 1985b), we could find no relationship between ir basic FGF content and the hormonal characteristics of the human tumours. The two tumours with ir basic FGF values which were much higher than the others were growth hormone or prolactin-secreting but there were no distinguishing features, either clinically or histologically, about these particular tumours. One of the characteristics of FGF is its strong affinity for heparin and in this respect ir FGF in pituitary extracts behaved in an identical manner to that of standard basic FGF, eluting maximally at 1.6-1.8~NaCl. In addition there was a smaller amount of material ~ This ir FGF has also been with a stronger affinity for heparin eluting at about 2 . 5 NaC1. reported by Baird et al. (1986) and is presumed to be another microheterogeneous form. The molecular form of ir basic FGF within the pituitary was found to be greatly different from that of authentic 1-146 FGF which has a molecular weight of approximately 16 kDa. Most of the ir basic FGF was present as a large molecular form and in the region of 70-150 kDa. Mormede et al. (1985) have reported similar high molecular weight forms of basic FGF in a variety of rat tissues including the pituitary gland of this species and they identified no FGF of 16 kDa molecular weight. The nature of the high molecular weight forms is not known, but it is significant that the glycosaminoglycan heparin to which FGF has a strong affinity is closely related to heparin sulphate (Kraemer, 1971) which is a component of the extracellular matrix of tissues. Recent work has shown that in vivo basic FGF binds strongly to glycosaminoglycans in the extracellular matrix of bovine endothelial cells (Baird & Ling, 1987) or the basement membrane-like matrix of a mouse sarcoma (Vigny et al., 1988). This extracellular binding can be prevented by degradation of the heparin sulphate chains with the enzyme heparitinase. It seems possible, therefore, that at least some of the high molecular weight forms of ir basic FGF found in pituitary tissue may represent binding to the glycosaminoglycans of the extracellular matrix. In view of the finding that FGF appears to be present extracellularly in many tissues, its site of origin within the pituitary requires consideration. Little or no basic FGF has been found in rat pituitary cell lines (Baird et al., 1986) and we have been unable to detect basic FGF either within cells or in the medium from cultures of a human prolactin-secreting tumour cell line (unpublished). Recently, Ferrara et al. (1987) have produced evidence that a separate population of pituitary cells, designated folliculo-stellate cells, are a major
Fibroblast growth factor in pituitary tumours
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source of basic FGF within the normal pituitary. Our finding of reduced amounts of ir basic F G F in tumorous pituitaries would be in agreement with this view since folliculostellate cells are not a predominant feature of adenomatous tissue. Whatever the site of origin, it is suggested by Baird and Ling (1987) that after its expression basic FGF would become associated with the glycosaminoglycans of the extracellular matrix or basement membrane to act as a reservoir for the growth factor. From this complex it would then be released by local enzyme action to act on specific cell surface F G F receptors to induce biological activity. The function of basic F G F in pituitary tumours, or indeed in normal pituitary glands, is not clear. However, recent work by us has shown that in contrast with its mitogenic activity on fibroblasts, basic 1-146 FGF inhibits the DNA synthesis of human pituitary tumour cells, whereas a different heparin-binding factor derived from pituitary glands is stimulatory to these same cells (Prysor-Jones et al., 1989). Schweigerer et al. (1987) have reported that basic F G F inhibits the growth of a human Ewing’s tumour cell line in culture but is stimulatory to some other tumours. It is clear, therefore, that the effect of basic F G F is complex since it can be stimulatory or inhibitory towards cell growth depending upon the particular cell type. The reason for this bifunctional effect is not known. We suggest, however, that in the case of pituitary tumours the reduction in ir basic F G F which we have demonstrated to be present in many instances would lead to less restraint of cell growth and the balance would then be tipped in favour of stimulatory factors. ACKNOWLEDGEMENTS
We are grateful to the Medical Research Council for financial support and to Dr A. Baird for antisera. REFERENCES BAIRD, A,, BOHLEN. P.,LING,N. & GUILLEMIN, R. (1985a) Radioimmunoassay for fibroblast growth factor (FGF): release by the bovine anterior pituitary in vitro. Regulatory Peptides, 10, 309-3 17. BAIRD,A., ESCH,F., MORMEDE, P., UENO,N., LING,N., BOHLEN,P., YING,S.Y., WEHRENBERG, W.B. & GUILLEMIN, R. (1986) Molecular characterization of fibroblast growth factor: distribution and biological activities in various tissues. Recent Progress in Hormone Research, 42, 143-205. BAIRD,A. & LING,N . (1987) Fibroblast growth factors are present in the extracellular matrix produced by endothelial cells in oitro. Biochemical and Biophysical Research Communications, 142,428-634. BAIRD, A., MORMEDE, P., YING,S.Y., WEHRENBERG, W.B., UENO,N., LING,N. &GUILLEMIN, R. (1985b)A nonmitogenic pituitary function of fibroblast growth factor: regulation of thyrotropin and prolactin secretion. Proceedings of National Academy of Sciences ofthe USA, 82, 554555549, BERTOLINI, J. & HEARN,M.T.W. (1987) Isolation, characterisation, and tissue localisation of an N-terminaltruncated variant of fibroblast growth factor. Molecular and Cellular Endocrinology, 51, 187-199. ESCH,F.,BAIRD, A., LING,N., UENO,N.,HILL,F.. DENEROY, L., KLEPPER, R., GOSFQDAROWICZ, D., BOHLEN, P. & GUILLEMIN, R. (1985) Primary structure of bovine pituitary fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Proceedings of National Academy of Sciences of the USA, 85,6507-651 1. FERRARA, N . , SCHWEIGERER, L.S., NEUFELD, G., MITCHELL, R. & GOSPODAROWITZ, D. (1987) Pituitary follicular cells produce fibroblast growth factor. Proceedings of National Academy ofsciences of the USA, 84, 5713-5711. GAMBARINI, A.G. & ARMELIN, H.S. (1982) Purification and partial characterization of an acidic fibroblast growth factor from bovine pituitary. Journal of Biological Chemistry, 257, 9692-9697.
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