Bromocriptine

Prolactinomas are one of the most frequent tumors of the human anterior pituitary. Dopamine agonists are the choice in the medical treatment of this disease. Bromocriptine (BC) is a well known anti-neoplasic agent in human PRL secreting adenomas although its effect on PRL cells is far from clear. We decided to investigate its influence on cell proliferation parameters: (3H)thymidine incorporation, expression of c-myc and c-fos, and number of estrogen receptors present in the samples. A total of 28 patients were included in this protocol They were treated with BC (5-7.5 mg day^sup -1^ patient^sup -1^) or with vehicle orally 15 days before surgery. We found that in BC treated patients (3H)thymidine incorporation was lower than in vehicle treated patients. The oncogenes expression were diminished in BC comparing with vehicle-treated patients. No difference in the number of estrogen receptors was observed in the samples from BC or vehicle-treated patients. These results clearly demonstrate that one mechanism to reduce the size of human PRL secreting adenomas by BC is the inhibition of DNA duplication. [Neurol Res 2001; 23: 721-723]

Keywords: Bromocriptine; prolactinomas; c-myc; c-fos

INTRODUCTION

Bromocriptine (BC), a dopamine agonist, is widely used in the treatment of hyperprolactinemia including pro(actin (PRO secreting adenomas. It reduces both the levels of serum PRL as well as the size of human and experimental prolactinomas1-4. In adult male rats when PRL release is stimulated with antidopaminergic drugs there is an increase in the proliferation of PRL secreting cells5 which is estrogen dependent6. When PRL release is inhibited proliferation of lactotrophs decreases. In adult rats BC inhibits synthesis and secretion of PRL by the anterior pituitary gland, reduces the incorporation of tritiated thymidine, the proliferation of PRL secreting cells3, the concentration of estradiol binding sites7 and the expression of the protooncogenes c-myc and c-fos8. BC also reduces the weight of the hyperplastic anterior pituitary gland in rats chronically treated with long acting estrogens3.

Prolactinomas are one of the most frequent tumors of the human anterior pituitary gland. Treatment is carried out medically with dopamine agonists or alternatively by surgery. The outcome of both treatments depends mainly on the tumor size and also on its response to the dopaminergic agonists.

We already know that c-myc and c-fos protooncogenes expression occurs in PRL secreting adenomas and that in these tumors there are estradiol binding sites9.

In this investigation we studied the effect of BC administered before surgery on the incorporation of tritiated thymidine to the tumor DNA as a tool to measure DNA synthesis. We also determined the levels of c-myc and c-fos mRNA and the concentration of estradiol binding sites in the prolactinomas after the administration of BC or vehicle.

MATERIALS AND METHODS

Patients

The patients studied had PRL secreting adenomas of grade II, III and IV according to the radiological classification10. Twenty-eight female patients aged 19 to 48 years old with PRL secreting adenomas of the anterior pituitary gland were studied. They all had hyperprolactinemia, and most of them galactorrhea and/ or amenorrhea.

They were treated with BC 5-7.5 mg day^sup -1^ patient^sup -1^ orally or with vehicle (similar pill without the active drug) for 15 days before surgery. Radiological tumor grades were equally distributed between vehicle and BC treated patients in the different determinations.

Tumor tissue (9-120 mg) were obtained by transeptoesphenoidal surgery one day after the last administration of BC and rapidly frozen in dry ice until estrogen receptor assay and/or c-myc and c-fos RNA isolation and purification was performed. Some of the tumors were very small and it was not possible to perform a complete study with them.

The two patients with grade IV had subfrontal surgery. Serum levels of PRL were determined by IRMA in 15 patients treated with vehicle (pre-treatment PRL levels 527.40 +/- 70.54 ng ml^sup -1^) and in 13 treated with BC (pretreatment PRL levels 591.90 +/- 78.66 ng ml^sup -1^) before surgery. There were no significant differences between the groups. Normal values 6-24 ng ml^sup -1^.

Determination of estrogen receptors

Studies on estrogen receptors were performed in tissue from eight patients (four-vehicle treated and four BC-- treated). The binding of (3H)estradiol was carried out in prolactinoma tissue removed at surgery as previously described7.

C-myc and c-fos studies

Expression of oncogenes were performed in tissue from 15 patients for c-myc (eight vehicle-treated and seven BC-treated) and in 10 patients for c-fos (five vehicle-treated and five BC-treated). Steady state levels of c-myc and c-fos mRNA was determined in total RNA obtained from the tumor tissue with guanidinium thiocyanate-phenol-chloroform as previously described8. RNA blotting (Northern) analysis: total RNA (20 yg) was denatured and separated by electrophoresis through a 1.5% agarose gel containing 18% formaldehyde and ethidium bromide. The RNA was transferred to nylon membranes, hybridized with the appropriate probes (c-myc: human third exon cloned in pBR322 and c-fos: murine genomic fragment 1.6 kb cloned in pSP65) labeled with 32PdCTP and submitted to autoradiography. Both probes were cloned, amplified by the polymerase chain reaction and labeled by Random Primer. Results are expressed as arbitrary units according to the densitometric tracings of the autoradiography. An 18s human ribosomal RNA probe was used to control the concentration of total RNA in the nylon membrane.

DNA synthesis

Adenomas from five BC- and six vehicle-treated patients were used to measure the incorporation of (3H)thymidine. Eight to 10 mg freshly obtained tissue were incubated for 30 min at 37 deg C under 95% O^sub 2^, 5% CO^sub 2^ in 0.5 ml medium TC199 (Difco, Detroit, MI, USA) in the presence of 2 uC(methyl-3H)thymidine (sp. act. 50.8 Ci mmol; New England Nuclear Co., Boston, MA, USA). The results were expressed as relative specific radioactivity which is (trichloroacetic acid insoluble disintegration/min per mg DNA)/(trichloroacetic acid soluble disintegrations/min per mg DNA)x1000. A detailed description of the incubation and further processing for the measurement of radioactivity has been described11.

Statistical analysis

Results are expressed as means +/- SEM. Statistical analysis was performed according to ANOVA.

Although this is not the purpose of this presentation, most of the patients are free of disease 2-8 years after surgery. Thyroid and adrenal axis were normal, while LH, FSH and estradiol were mostly low before BC administration and 62% of the patients normalize these values after BC administration and before surgery.

DISCUSSION

Prolactinomas are a heterogenous group of tumors in the anterior pituitary gland. In this study we are comparing adenomas from different patients and consequently with different biological behavior. However, the results clearly indicate differences between vehicle- and BC-- treated patients indicating that BC do have an inhibitory effect in several parameters related to DNA synthesis and PRL cell proliferation. This is in accordance with a number of clinical observations reporting important reductions in the size of PRL secreting adenomas after the administration of BC to the patients1,4,12.

Previous studies from our group in adult rats showed that BC inhibits DNA synthesis and the expression of the protooncogenes c-myc and c-fos3,8 In these animals the reduction in the steady state levels of c-myc and c-fos mRNA produced by BC precedes to the inhibition of DNA synthesis and cell proliferation in PRL secreting cells.

Since BC binds to the D2 receptor at the cell membrane and c-myc and c-fos proteins are transcription factors in the nucleus of the cell, the information to induce these modifications might involve a cascade of events to carry the signal from the receptor to the cell genome. Whether the inhibition of DNA synthesis and the expression of both protooncogenes by BC are independent events or they are associated, remains to be studied. D2 receptor inactivates in PRL secreting cells the c-AMP and the phosphatidyl inositol cascades.

We have previously shown in the rat anterior pituitary gland a correlation between incorporation of tritiated thymidine and PRL cell proliferation as determined by the number of mitosis13. In the pituitary gland of these animals there is a close relationship between PRL secretion and DNA replication. Prolactinomas, as other pituitary adenomas, are monoclonal but the genetic alteration of the PRL secreting cell is not known at this time. No amplifications of c-myc and c-fos have been found in our laboratory (unpublished observation) and so far no mutations or rearrangements have been described.

Although the concentration of estradiol binding sites in the anterior pituitary gland of the rat are reduced after the administration of BC7 we did not find significant changes in human prolactinomas. Perhaps the number of studies performed is too low to draw a definitive conclusion regarding this parameter. The scarcity of the material obtained in human prolactinomas makes it difficult to increase the number of determinations.

The results of these studies clearly demonstrate that one mechanism to reduce the size of human PRL secreting adenomas produced by BC is the inhibition of DNA replication, because (3H)thymidine incorporation as well as expression of c-myc and c-fos are decreased.

ACKNOWLEDGMENTS

These studies were supported by grants from Agencia National para la Promotion Cientifica y Tecnologica BID 802/OC-AR-PICT No 05-- 3406, and by CONICET.

REFERENCES

1 Bergh T, Nillius SJ, Wilde L. Bromocriptine treatment of 42 hyperprolactinaemic women with secondary amenorrhoea. Acta Endocrinologica 1978; 88: 435-451

2 Friesen MG, Tolls G. The use of bromocriptine in the galactorrhoea syndromes: The Canadian cooperative study. Clin Endocrinol 1977; 6: 915-995

3 Kalbermann LE, Machiavelli GA, De Nicola AF, Weissenberg LS, Burdman JA. Synthesis of DNA in oestrogen-induced pituitary tumours in rats: Effect of bromocriptine. J Endocrinol 1980; 87: 221-224

4 Thorner MO, Martin WH, Rogol AD, Morris JL, Perryman RL, Conway BP, Howards SS, Wolfman MG, MacLeod RM. Rapid regression of pituitary prolactinomas during bromocriptine treatment. J Clin Endocrinol Metab 1980; 51: 438-445

5 Kalbermann LE, Szijan I, Burdman JA. Cell proliferation in the rat pituitary gland. A mechanism of control in prolactin cells. Experientia 1979; 35: 689-690

6 Burdman JA, Calabrese MT, Romano MI, Carricarte VC, MacLeod RM. Effect of ovariectomy and clomiphene on the in vitro incorporation of (3H)thymidine into pituitary DNA and on prolactin synthesis and release in rats. J Endocrinol 1984; 101: 197-200

7 Szijan I, Burdman JA, Alonso GE. Effects of dopamine agonists and antagonists on the binding of (3H)estradiol to its receptors in the anterior pituitary gland of male rats. Endocrinology 1985; 117: 1742-1744

8 Chernavsky AC, Valerani AV, Burdman JA. Haloperidol and estrogens induce c-myc and c-fos expression in the anterior pituitary gland of the rat. Neurol Res 1993; 15: 339-342

9 Machivelli GA, Rivolta CM, Aretese R, Basso A, Burdman JA. Expression of c-myc and c-fos and binding sites for estradiol and progesteron in human pituitary tumors. Neurol Res 1998; 20: 709-712

10 Sutton TJ, Vezina JL. Co-existing pituitary adenoma and intrasellar arachnoid invagination. Am J Roentgenol Radium Ther Nucl Med 1974; 120:46-54

11 Kalbermann LE, Szijan I, Jahn GA, Krawiec L, Burdman JA. DNA synthesis in the pituitary gland of the rat. Effect of sulpiride and postnatal maturation. Neuroendocrinology 1979; 29: 42-48

12 McGregor AM, Scanlon MF, Hall R, Hall K. Effects of bromocriptine on pituitary tumour size. Brit Med J 1979; 11: 700-703

13 Perez RL, Machiavelli GA, Romano MI, Burdman JA. Prolactin release, oestrogens and proliferation of prolactin secreting cells in the anterior pituitary gland of adult male rats. J Endocrinol 1986; 108:399-401

lose A. Burdman*^ ^^ sec, Liliana N. Guerra* para, Maria T. Calabrese and Armando Basso^^

*Department of Endocrinology, Centro de Investigaciones Medicas Albert Einstein - Fundacion CIMAE

^Servicio de Endocrinologia, Hospital Israelita 'EZRAH'

^^Catedra de Neurocirugia, Hospital de Clinicas Jose de San Martin, Faculty of Medicine sec Universidad Abierta Interamericana, Faculty of Medicine, Buenos Aires

para University of Buenos Aires, Faculty of Exacts and National Sciences, Argentina deg

Correspondence and reprint requests to: Dr J.A. Burdman, Fundacion CIMAE, Luis Viale 2831, (1416) Buenos Aires, Argentina. [jaburdman@fibertel.com.ar] Accepted for publication March 2001.

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