Estrogen receptors in human pituitary tumors.

The relationship between the presence of estrogen receptors in pituitary adenomas and the post surgical evolution of the patients in order to find another prognostic parameter for these tumors have been studied to improve the treatment selection. Estrogen receptors were studied by immunocytochemistry in histological sections of paraffin embedded 42 pituitary adenomas. Only 19% of these tumors were positive for estrogen receptors. As expected, the higher concentration (60%) was found in prolactin secreting adenomas, although we found estrogen receptors in one somatotroph and in one nonsecreting. The most aggressive tumors were those negative for estrogen receptors within the prolactinomas and the positive somatotrophinoma. The results of this work suggest that the determination of estrogen receptors in pituitary tumors might help as a prognostic factor in these adenomas.

Telomerase activity in fine needle aspiration biopsy samples: application to diagnosis of human thyroid carcinoma.

BACKGROUND: The diagnosis of thyroid follicular carcinoma by fine needle aspiration biopsy is a well known problem in thyroid pathology. METHODS: We evaluated telomerase activity (TA) in 85 fine needle aspiration biopsy (FNAB) samples from patients with thyroid nodules. Surgery samples from patients with tumor or follicular adenomas were also analyzed. RESULTS: Twenty of the FNAB samples corresponded to carcinomas and were positive to telomerase assay (TA >10 Units). Among them, 4 follicular carcinomas and 1 papillary carcinoma were labeled as indeterminate by FNAB cytological examination. Four percent false positive cases and no false negative cases for TA in FNABs were reported. FNAB samples from follicular adenomas were diagnosed as indeterminate by cytological examination, but they showed no detectable TA. Tumor tissues from patients with follicular or papillary thyroid carcinomas presented TA >10 Units, whereas follicular adenoma tissues (benign nodules) showed no TA. CONCLUSION: Our results showed a good correlation between TA in FNAB samples and tumor/nodule thyroid tissue. This suggested that use of TA as a biological marker of malignancy might be a useful tool in the diagnosis of follicular thyroid carcinomas or follicular thyroid adenomas using FNAB samples.

Bromocriptine and the expression of c-myc and c-fos in human prolactinomas.

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 (5-7.5mg day(-1) patient(-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.

Antioxidants in the treatment of Graves disease.

An antioxidant mixture (LAROTABE) was evaluated in the treatment of Graves disease. Fifty-six hyperthyroid patients were treated with methimazol (MMI) (A), LAROTABE (B), or MMI plus LAROTABE (C). According to a clinical score, improvement was obtained at 8 weeks in A and 4 weeks in B and C. Group A diminished their thyroid hormone concentration to normal levels, while patients with LAROTABE did not reduce T3 and T4 unless MMI was introduced. Hyperthyroid patients had increased malondialdehyde (MDA) content and SOD activity and decreased catalase activity compared to controls. Within group A, MDA decreased to control values while SOD was reduced 38.3% and catalase increased 21.6%. Similar results were obtained for MDA and for both enzymes after treatment with LAROTABE. Signs and symptoms of Graves disease might be related to an increase in free radicals; antioxidants could be a new therapeutic tool to improve the clinical manifestation of this illness.

Alpha subunit of glycoprotein hormones in the sera of acromegalic patients and its mRNA in the tumors.

Within a population of 16 pituitary adenomas we found high levels of glycoprotein alpha subunits in the sera of patients with somatotrophic tumors. This finding was correlated with the presence of mRNA alpha subunit in these tumors indicating the adenomas themselves as the origin of the circulating alpha-subunit. Synthesis of these two hormones, which are chemically very different, by the same tumor cells indicates a high degree of differentiation of these cells. We are unable at this time to conclusively correlate differentiation of these tumors aggressively.

Expression of c-myc and c-fos and binding sites for estradiol and progesterone in human pituitary tumors.

We studied the concentration of mRNA from the oncogenes c-myc and c-fos in human pituitary adenomas by Northern blot hybridization (35 somatotrophinomas, 9 prolactinomas, 21 nonsecreting and 3 adrenocorticotrophinomas). The concentration of estrogens and progesterone receptors was also investigated. The levels of c-myc and c-fos mRNA was higher in nonsecreting tumors which were generally the largest and had a higher percentage of recurrence after surgery than the other groups. High concentration of estrogen receptors was observed in tumors derived from cells which are normally the target of this hormone, mainly prolactinomas. They were also present in somatotrophic and nonsecreting adenomas, related to the presence of prolactin or gonadotrophin cells in these tumors. The presence of estrogen receptors indicates that the tumor cells maintain their differentiation and a good prognosis as is the case for prolactinomas. We did not find any relationship between estrogen receptors and the concentration of c-myc and c-fos oncogenes. Larger adenomas (mainly nonsecreting) had higher levels of c-myc and c-fos mRNA than the other tumors and they had an important percentage of recurrence after surgery. It is clear that tumor size is related to the outcome after surgery and that nonsecreting adenomas are usually large because of the late diagnosis. However two large somatotrophinomas with extrasellar expansion also had overexpression of both oncogenes and both relapsed after surgery.

Pituitary tumors in adolescent patients.

We report on a series of 48 patients, ages 14 to 20 year, with hypophyseal adenomas. Of these, 46 (96%) had secreting tumors, 3 had Cushing's disease, 9 had somatotrophinomas, and 34 (29 females and 5 males) had prolactinomas. Thirty cases were diagnosed as intrasellar adenomas (62%) while the remaining eighteen (38%) presented extrasellar expansion. Of 9 acromegalic patients, 7 had typical clinical and biochemical features 2 were exclusively prognatic with normal basal GH levels, but abnormal dynamic tests. Prolactinomas were noninvasive in women and faster growing and more extensive in men. Forty seven patients underwent surgery. Five of these required craniotomy and the rest approached through the sphenoidal bone (TSE). Remission was achieved in Cushing's disease, acromegaly, and female intrasellar prolactinomas. Larger tumors such as nonsecreting adenomas and male prolactinomas showed poor results after undergoing subtotal resections, with persistence of endocrinological disturbances. From our findings it appears that these tumors are aggressive in youth than in adults. Because there was a close relationship between tumor size, invasiveness, and the patients' final outcome, we conclude that early diagnosis and treatment is essential. Frequent complaints in adolescents such as irregular menses, retarded puberty, and growth disorders should be thoroughly investigated and not merely considered as transient or 'functional'.

Ultrastructural modifications and changes in the expression of hormonal genes produced by indomethacin in the anterior pituitary gland of the rat.

Indomethacin decreases the level of prolactin (50%) and growth hormone (70%) mRNA in the anterior pituitary gland of the rat. Actin mRNA increases (59%). Ultrastructurally there is a decrease in the number of secretory granules. Indomethacin also prevents the increase in prolactin secretory granules produced by the administration of estradiol. The results indicate that indomethacin inhibits hormonal synthesis in the APG at a transcriptional level. This effect appears selective because mRNA level for actin synthesis in the pituitary gland was higher than in nontreated rats.

Haloperidol and oestrogens induce c-myc and c-fos expression in the anterior pituitary gland of the rat.

Decreased dopaminergic and increased oestrogenic effects induce prolactin release and DNA synthesis in prolactin secreting cells of the adult male rats. Oestrogen treatment for 7 days significantly increased the levels of prolactin, c-myc and c-fos mRNAs. The effect of oestrogens was reversed by the administration of the dopaminergic agonist bromocriptine. There was an early gradual increase of c-myc mRNA levels beginning 30 min after the injection of the steroid. c-fos mRNA levels increased sharply 15 min after oestrogen administration and decreased to basal values 15 min later to remain at this level over the period of time evaluated. Administration of the dopaminergic antagonist haloperidol did not change the levels of protooncogenes mRNA. However, in rats previously treated with oestrogens for 7 days c-myc mRNA levels increased 90 min after the injection of haloperidol and decreased to basal values after 2.5 h. c-fos mRNA levels increased sharply 30 min after haloperidol administration and also decreased to basal values 1 h later. We propose that changes in protooncogenes expression may be involved in the stimulation of cell proliferation induced by prolactin secretion.

Human pituitary tumours: studies on genes expression.

Searching for differences in gene expression between different types of human pituitary adenomas, we evaluated the concentration of mRNA from hormonal genes (prolactin and growth hormone) and oncogenes (c-myc and c-fos) in 12 growth hormone-secreting 7 prolactin-secreting and 11 nonsecreting adenomas. We found a positive correlation between clinical diagnoses and hormonal gene expression in all the cases. Our reports indicate the presence of c-myc and c-fos mRNA in all the adenomas evaluated. The concentration of c-myc mRNA was higher in somatotrophic adenomas than in prolactinomas and nonsecreting adenomas whereas c-fos mRNA concentration was similar in the different types of tumours analysed. Oncogenes products, in turn, might stimulate DNA synthesis and cell proliferation and eventually lead to the formation of pituitary adenomas. This is a working hypothesis.

Relationship between the level of c-myc mRNA and histologic aggressiveness in thyroid tumors.

We investigated the relationship between the level of c-myc mRNA and histologic aggressiveness in thyroid tumors obtained at surgery. In thyroid carcinomas, there was a positive correlation between these two parameters, while in benign tumors there was no correlation between cellularity and the expression of the proto-oncogene c-myc. These results might be useful in the prognosis of thyroid tumors and consequently helpful in the management of the patient.

Indomethacin inhibits the effects of oestrogen in the anterior pituitary gland of the rat.

Two inhibitors of prostaglandin synthesis, indomethacin and aspirin, blocked the increase of oestrogen-binding sites in the nuclear subcellular fraction, an increase which occurs after the administration of oestradiol. Consequently the biological effects of oestrogens in the anterior pituitary gland of the rat (prolactin synthesis, concentration of progesterone-binding sites and cell proliferation) are diminished. The anterior pituitary gland synthesized prostaglandin F2 alpha (PGF2 alpha), PGE2 and PGD2 from arachidonic acid. This synthesis was blocked when indomethacin was added to the culture media. Oestrogen increased the concentration of PGE2: an increase that was partially prevented by indomethacin. Prostaglandins may have an important role on the effects of oestrogen in the anterior pituitary gland of the rat.

Prolactin release, oestrogens and proliferation of prolactin-secreting cells in the anterior pituitary gland of adult male rats.

Relationships among the release of prolactin, the effect of oestrogens and the proliferation of prolactin-secreting cells were studied under several experimental conditions. Administration of sulpiride or oestradiol released prolactin and stimulated cell proliferation in the anterior pituitary gland of adult male rats. Clomiphene completely abolished the rise in cell proliferation, but did not interfere with the sulpiride-induced release of prolactin. Treatment with oestradiol plus sulpiride significantly increased serum prolactin concentrations and the mitotic index compared with the sum of the stimulation produced by both drugs separately. Bromocriptine abolished the stimulatory effect of oestradiol on the serum prolactin concentration and on cell proliferation. In oestradiol- and/or sulpiride-treated rats, 80% of the cells in mitoses were lactotrophs. The remaining 20% did not stain with antisera against any of the pituitary hormones. The number of prolactin-secreting cells in the anterior pituitary gland significantly increased after the administration of oestradiol or sulpiride. The results demonstrate that treatment with sulpiride and/or oestradiol increases the proliferation and the number of lactotrophs in the anterior pituitary gland of the rat.

Relationship between release of LH and incorporation of tritiated thymidine in the anterior pituitary gland of the castrated male rat: effect of LHRH and its highly active analogue buserelin.

Castration of the adult male rat significantly (P less than 0.01) increased the concentration of LH in serum and the incorporation of (3H) thymidine into the pituitary DNA. The administration of a single dose of LHRH or its analogue buserelin stimulated the release of LH but it did not modify (3H) thymidine incorporation. When multiple doses of LHRH or buserelin were injected, there was a significant (P less than 0.01) inhibition of LH release and also the incorporation of (3H) thymidine into the DNA of the anterior pituitary gland was significantly (P less than 0.01) diminished. These observations are compatible with the idea of the close relationship between hormonal release and DNA synthesis in the anterior pituitary gland of the rat.

Effect of dopamine agonists and antagonists on the binding of [3H]estradiol to its receptors in the anterior pituitary gland of male rats.

The synthesis of PRL and DNA in PRL cells is regulated by estrogens and dopamine. To investigate a possible relationship between these two components, we studied the influence of dopamine agonists and antagonists on the binding of [3H]estradiol ([3H]E2) to its receptors in the anterior pituitary gland of estrogenized male rats. The administration of sulpiride (dopamine antagonist) or bromocriptine (dopamine agonist) decreased the binding of [3H]E2 to cytosolic receptors when the concentration of [3H]E2 in the assay mixture was 1 nM. Both drugs also diminished the binding of [3H]E2 when they were added in vitro to the incubation media, apparently in a competitive way. Dopamine and alpha-methyltyrosine also inhibited competitively the binding of [3H]E2 to cytosolic receptors. The inhibition constants were determined by the Lineweaver-Burk plot. To overcome the competitive inhibition of dopamine agonists and antagonists, the concentration of the titrated steroid in the incubation mixture was increased to 16 nM. This concentration was established by saturation analysis. The administration of alpha-methyltyrosine increased the binding of [3H]E2 to nuclear receptors without modifying the binding to cytosolic receptors. This increase paralleled an increment in the levels of plasma PRL. Bromocriptine prevented the increase in [3H]E2 binding produced by alpha-methyltyrosine and had no effect on the binding when administered to nontreated rats. These results suggest that dopamine can regulate the biological effects of estradiol in the anterior pituitary gland by decreasing the binding of this hormone to its receptors.

Spontaneous sparsely-granulated prolactin-producing pituitary adenomas in aging rats. A prospective study of the effect of bromocriptine.

The effect of bromocriptine (BEC) treatment on spontaneous, sparsely granulated, prolactin-producing pituitary adenomas was studied in aging female Long-Evans rats of at least 23 months of age. Rats treated with BEC for 1-44 days showed a marked decrease in serum prolactin (PRL) concentrations at the end of the treatment period (9.1-34 ng/ml) when compared to the serum PRL levels of age-matched control animals (94.6-233 ng/ml). No significant differences in serum PRL levels (ng/ml; mean +/- SEM) were noted in rats withdrawn for 14 days from BEC treatment (132.9 +/- 18.8) when compared to age-matched controls (181.5 +/- 70.9). The mean pituitary weight (mg) was significantly reduced in the rats treated for 44 days with BEC (23.4 +/- 1.4) compared to untreated controls (43.4 +/- 8.3). At the time of sacrifice, PRL-producing adenomas were found in 16 of 33 control rats, 5 of 10 rats treated for 1 day with BEC, 5 of 20 rats treated with BEC for 44 days, and 12 of 28 rats in the animals withdrawn from BEC treatment for 14 days. Morphometric analysis of sparsely granulated PRL-containing adenomas revealed that, although the nuclear area was reduced after 1 day of BEC treatment, the cytoplasmic area was reduced only after 44 days. Forming granule diameters were significantly increased after 44 days of BEC treatment and markedly decreased in the withdrawal group. Storage granule diameters were increased in both the 1-day and 44-day groups and were decreased in rats withdrawn from BEC for 14 days. Rough endoplasmic reticulum, forming granule, storage granule, and lysosome volume densities were increased after 1 day of BEC treatment. The Golgi region volume density decreased only after 44 days of BEC treatment. We conclude that aging female Long-Evans rats harboring PRL-producing pituitary adenomas can respond to BEC administration with a decrease in serum PRL levels and morphologic changes in adenoma cells. However, the structural alterations in PRL cells of the rat adenomas are less conspicuous than those of human tumors. In the rat, like in human patients, a direct toxic effect of BEC on PRL-producing adenoma cells has not been demonstrated.

Relationship between release of LH and incorporation of tritiated thymidine in the anterior pituitary gland of the castrated female rat.

Castration of female rats during diestrus increases the concentration of circulating LH from days 3 and the incorporation of 3H thymidine into pituitary DNA from day 5. Both effects are completely abolished by the administration of dihydrotestosterone. Although the i.v. injection of LHRH markedly enhances the concentration of LH in serum, it does not modify the incorporation of 3H thymidine into pituitary DNA. Castration might produce a maximal stimulation in 3H thymidine incorporation and a further stimulation of LH release with LHRH is unable to enhance the incorporation of the radioactive precursor. The results suggest a relationship between LH secretion and DNA synthesis in the pituitary gland of the rat.

Correlation between LH secretion in castrated rats with cellular proliferation and synthesis of DNA in the anterior pituitary gland.

The relationship between the release of LH and the synthesis of DNA was studied in the anterior pituitary gland of castrated rats. Cell types were characterized immunocytochemically. Castration significantly (P less than 0.01) increased the concentration of LH in serum (1326%) and the incorporation of [3H]thymidine into pituitary DNA (72%). This was accompanied by an increment in the activity of the enzyme DNA polymerase-alpha (58%) and in the number of mitoses (from 2 +/- 0.1/mm2 in intact rats to 21 +/- 0.8/mm2 15 days after castration). Only 20% of the mitoses found in the pituitary gland of castrated rats were positively stained with the antiserum against the beta-subunit of LH. The other 80% did not stain either with LH antiserum or with antisera against the other pituitary hormones. There was a significant (P less than 0.01) increase in the number of LH cells in castrated rats (48%). All the changes produced in the anterior pituitary gland after castration were prevented by the administration of dihydrotestosterone. The results demonstrate that a stimulation of LH release is followed by an increase of DNA synthesis and cell proliferation of gonadotrophs in the anterior pituitary gland.

Effect of ovariectomy and clomiphene on the in-vitro incorporation of [3H]thymidine into pituitary DNA and on prolactin synthesis and release in rats.

In the anterior pituitary gland changes in prolactin synthesis and in the incorporation of [3H]thymidine into DNA are coincident under several experimental conditions. We investigated whether these changes are obligatory, thus indicating a regulatory mechanism common to the synthesis of both macromolecules. Alternatively, the parallel changes may represent similar responses to various stimuli operating through different pathways. The administration of alpha-methyl-p-tyrosine (alpha MpT) to rats stimulated the incorporation of [3H]leucine into prolactin and [3H]thymidine into DNA. When the effectiveness of oestrogen was suppressed by ovariectomy or by blockage of oestrogen receptors by the antioestrogen clomiphene, alpha MpT stimulated the synthesis of prolactin but not the incorporation of [3H]thymidine into pituitary DNA. The results clearly indicate two independent mechanisms regulating the synthesis of prolactin and DNA in the anterior pituitary gland.

Regulation of pituitary DNA synthesis during different reproductive states in the female rat: role of estrogens and prolactin.

We studied DNA synthesis in the rat adenohypophysis during the estrous cycle, pregnancy and lactation. During the estrous cycle, DNA synthesis was 3 times higher on the morning of estrus than on the other days. This peak was abolished completely by ovariectomy or pentobarbital, which also blocked the preovulatory surges of LH and prolactin. Methallibure , which blocked the LH but not the prolaction surge, had a partial effect on DNA synthesis. An acute and significant decrease in pituitary DNA synthesis occurred between days 0 (estrus) and 1 of pregnancy, followed by a less pronounced diminution until parturition. After delivery, DNA synthesis increased steeply on day 1 of lactation, returning to low values by day 3, under normal suckling conditions. Thelectomy , which blocked suckling-induced prolactin release, or antiestrogen treatment, which did not decrease prolactin secretion, diminished pituitary DNA synthesis on day 1 of lactation. Estrogen administration to intact or ovariectomized rats on days 9-11 of lactation stimulated (100%) DNA synthesis. Ovariectomy had no effect. In conclusion, in the different reproductive states studied, pituitary DNA synthesis is related to prolactin release in the presence of estrogens.