0021-972X/91/7203-0687$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society
Vol. 72, No. 3 Printed in U.S.A.
Preliminary Characterization of Growth Factors Secreted by Human Pituitary Tumors JONATHAN WEBSTER*, JACK HAM, JOHN S. BEVAN, CAROLA D. TEN HORNf, AND MAURICE F. SCANLON Neuroendocrine Unit, Department of Medicine, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, Wales, United Kingdom
ABSTRACT. To investigate the secretion of mitogenic factors by human pituitary tumors we have cultured cells from 54 adenomas in serum-free medium. Conditioned media from 28 (52%) elicited dose-dependent stimulation of [3H]thymidine incorporation into rat GH3 cells (22-338% above control), while 14 (26%) inhibited GH3 proliferation. Stimulating activity was observed more frequently in nonfunctional tumor-conditioned medium (73%; n = 22) than in secretory tumor-conditioned medium (37%; n = 32). Of 10 tumour-conditioned media with mitogenic activity for GH3 cells, only 4 produced modest stimulation of HEp2 (human laryngeal carcinoma) cells. In contrast, [3H]thymidine incorporation into A431 (human squamous carcinoma) and PC12 (rat adrenal pheochromocytoma) cells was enhanced by each of 15 tumor-conditioned media (up to 342% and 275%, respectively), 8 of which had shown stimulatory and
2 inhibitory effects on GH3 cells. Gel filtration of pooled conditioned media from 10 nonfunctional tumors showed significant growth-promoting activity for GH3 cells in fractions corresponding to mol wt of 2-3 and 11-18 kDa. Proliferative activity on A431 cells also eluted in two positions; one corresponded to the higher mol wt peak seen with GH3 cells, while the other, not observed with GH3 cells, was in the 3- to 6-kDa range. These findings suggest that cells derived from human pituitary adenoma tissue synthesize and secrete several growth factors, each of which may have its own target cell specificities. These factors have yet to be characterized, but we suggest that they may have a role in stimulating the development or maintenance of human pituitary adenomas. (J Clin Endocrinol Metab 72: 687-692, 1991)
T
HE MOLECULAR basis of the development of human pituitary tumors is unknown. Recent evidence,.including the demonstration of a monoclonal population of cells in gonadotroph and somatotroph adenomas and of transforming activity in transfected DNA from a somatotroph and a nonfunctioning adenoma suggests that at least some may arise as a result of somatic mutation (1). Landis et al. (2) demonstrated activating point mutations in the a-subunit of the regulatory protein Gs in about 30% of GH-secreting pituitary tumors, which may account for the abnormal secretion and growth in this tumor subgroup. Loss of growth inhibitory influences at either the growth factor (3) or receptor level (4) may also have a role in pituitary tumor development. Another mechanism by which mutation may lead to uncontrolled growth is altered expression of autocrine growth-promoting factors. Such autocrine stimulation has been clearly demonstrated in a large number of malignant tumours and cell lines, including the PRLand GH-secreting rat pituitary tumor cell line GH3 (5,
6), a n d has recently been described in benign t h y r o i d
follicular adenomas, which produce and respond to insulin-like growth factor-I (IGF-I). (7). The normal anterior pituitary gland is known to be a source of several growth factors, including IGF-I (8, 9), IGF-II (10), epidermal growth factor (EGF) (11), basic fibroblast growth factor (bFGF) (12), transforming growth factor-a (13), and nerve growth factor (14), and at least some of these may be involved in the paracrine modulation of hormone secretion and possibly also cell turnover (15). Some human pituitary adenomas have been shown to secrete a chondrocyte growth factor (16). Others may produce as yet uncharacterized substances capable of transforming mouse fibroblasts (17). However, it is not yet certain whether these or other factors produced by human pituitary tumors have autocrine activity. We have, therefore, investigated the secretion of mitogenic substances by human pituitary tumor cells and report data concerning their target cell specificity and initial characterization.
Received July 2,1990. * Medical Research Council Training Fellow. To whom requests for reprints should be addressed. t Visiting Research Fellow from the Department of Endocrinology, University Hospital of Utrecht, Utrecht, The Netherlands.
Materials and Methods All tissue culture materials and reagents were purchased from either Gibco Ltd. (Paisley, Scotland) or Flow Laboratories 687
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 03:00 For personal use only. No other uses without permission. . All rights reserved.
WEBSTER ET AL.
688
(Rickmansworth, Hertfordshire, England). Radiochemicals were obtained from Amersham UK, and dialysis membranes from Medicel International Ltd. (London, England). All other chemicals, except where stated, were obtained from either Sigma Chemical Co. Ltd. (Poole, England) or BDH Ltd. (Bristol, England). Cell lines GH3 cells (18) were maintained in Ham's F-10 medium supplemented with 15% horse serum, 2.5% fetal calf serum, 2 mmol/L glutamine, and antibiotics (100 U/mL benzyl penicillin, 100 mg/L streptomycin, and 2.5 mg/L amphotericin-B). A431 (human squamous carcinoma) (19), HEp2 (human laryngeal carcinoma) (20), and PC12 cells (rat adrenal pheochromocytoma) (21) were grown in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum and antibiotics as described above. All cultures were maintained at 37 C in the presence of 5% CO2 and subcultured as required. Pituitary tumor cell cultures Human tumor tissue was obtained from three centers: Cardiff, Oxford, and Glasgow. Tissue from the latter two was transported by same day or overnight delivery in serum-free Minimum Essential Medium (MEM) D-valine supplemented with transferrin (0.12 /imol/L), dexamethasone (5 nmol/L), insulin (0.8 ^mol/L), T3 (30 pmol/L), sodium selenite (20 nmol/ L), and antibiotics as described above. Tissue was diced (
Vol. 72, No. 3 Printed in U.S.A.
Preliminary Characterization of Growth Factors Secreted by Human Pituitary Tumors JONATHAN WEBSTER*, JACK HAM, JOHN S. BEVAN, CAROLA D. TEN HORNf, AND MAURICE F. SCANLON Neuroendocrine Unit, Department of Medicine, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, Wales, United Kingdom
ABSTRACT. To investigate the secretion of mitogenic factors by human pituitary tumors we have cultured cells from 54 adenomas in serum-free medium. Conditioned media from 28 (52%) elicited dose-dependent stimulation of [3H]thymidine incorporation into rat GH3 cells (22-338% above control), while 14 (26%) inhibited GH3 proliferation. Stimulating activity was observed more frequently in nonfunctional tumor-conditioned medium (73%; n = 22) than in secretory tumor-conditioned medium (37%; n = 32). Of 10 tumour-conditioned media with mitogenic activity for GH3 cells, only 4 produced modest stimulation of HEp2 (human laryngeal carcinoma) cells. In contrast, [3H]thymidine incorporation into A431 (human squamous carcinoma) and PC12 (rat adrenal pheochromocytoma) cells was enhanced by each of 15 tumor-conditioned media (up to 342% and 275%, respectively), 8 of which had shown stimulatory and
2 inhibitory effects on GH3 cells. Gel filtration of pooled conditioned media from 10 nonfunctional tumors showed significant growth-promoting activity for GH3 cells in fractions corresponding to mol wt of 2-3 and 11-18 kDa. Proliferative activity on A431 cells also eluted in two positions; one corresponded to the higher mol wt peak seen with GH3 cells, while the other, not observed with GH3 cells, was in the 3- to 6-kDa range. These findings suggest that cells derived from human pituitary adenoma tissue synthesize and secrete several growth factors, each of which may have its own target cell specificities. These factors have yet to be characterized, but we suggest that they may have a role in stimulating the development or maintenance of human pituitary adenomas. (J Clin Endocrinol Metab 72: 687-692, 1991)
T
HE MOLECULAR basis of the development of human pituitary tumors is unknown. Recent evidence,.including the demonstration of a monoclonal population of cells in gonadotroph and somatotroph adenomas and of transforming activity in transfected DNA from a somatotroph and a nonfunctioning adenoma suggests that at least some may arise as a result of somatic mutation (1). Landis et al. (2) demonstrated activating point mutations in the a-subunit of the regulatory protein Gs in about 30% of GH-secreting pituitary tumors, which may account for the abnormal secretion and growth in this tumor subgroup. Loss of growth inhibitory influences at either the growth factor (3) or receptor level (4) may also have a role in pituitary tumor development. Another mechanism by which mutation may lead to uncontrolled growth is altered expression of autocrine growth-promoting factors. Such autocrine stimulation has been clearly demonstrated in a large number of malignant tumours and cell lines, including the PRLand GH-secreting rat pituitary tumor cell line GH3 (5,
6), a n d has recently been described in benign t h y r o i d
follicular adenomas, which produce and respond to insulin-like growth factor-I (IGF-I). (7). The normal anterior pituitary gland is known to be a source of several growth factors, including IGF-I (8, 9), IGF-II (10), epidermal growth factor (EGF) (11), basic fibroblast growth factor (bFGF) (12), transforming growth factor-a (13), and nerve growth factor (14), and at least some of these may be involved in the paracrine modulation of hormone secretion and possibly also cell turnover (15). Some human pituitary adenomas have been shown to secrete a chondrocyte growth factor (16). Others may produce as yet uncharacterized substances capable of transforming mouse fibroblasts (17). However, it is not yet certain whether these or other factors produced by human pituitary tumors have autocrine activity. We have, therefore, investigated the secretion of mitogenic substances by human pituitary tumor cells and report data concerning their target cell specificity and initial characterization.
Received July 2,1990. * Medical Research Council Training Fellow. To whom requests for reprints should be addressed. t Visiting Research Fellow from the Department of Endocrinology, University Hospital of Utrecht, Utrecht, The Netherlands.
Materials and Methods All tissue culture materials and reagents were purchased from either Gibco Ltd. (Paisley, Scotland) or Flow Laboratories 687
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 03:00 For personal use only. No other uses without permission. . All rights reserved.
WEBSTER ET AL.
688
(Rickmansworth, Hertfordshire, England). Radiochemicals were obtained from Amersham UK, and dialysis membranes from Medicel International Ltd. (London, England). All other chemicals, except where stated, were obtained from either Sigma Chemical Co. Ltd. (Poole, England) or BDH Ltd. (Bristol, England). Cell lines GH3 cells (18) were maintained in Ham's F-10 medium supplemented with 15% horse serum, 2.5% fetal calf serum, 2 mmol/L glutamine, and antibiotics (100 U/mL benzyl penicillin, 100 mg/L streptomycin, and 2.5 mg/L amphotericin-B). A431 (human squamous carcinoma) (19), HEp2 (human laryngeal carcinoma) (20), and PC12 cells (rat adrenal pheochromocytoma) (21) were grown in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum and antibiotics as described above. All cultures were maintained at 37 C in the presence of 5% CO2 and subcultured as required. Pituitary tumor cell cultures Human tumor tissue was obtained from three centers: Cardiff, Oxford, and Glasgow. Tissue from the latter two was transported by same day or overnight delivery in serum-free Minimum Essential Medium (MEM) D-valine supplemented with transferrin (0.12 /imol/L), dexamethasone (5 nmol/L), insulin (0.8 ^mol/L), T3 (30 pmol/L), sodium selenite (20 nmol/ L), and antibiotics as described above. Tissue was diced (
