Gene therapy in the neuroendocrine system.

The implementation of experimental gene therapy in animal models of neuroendocrine diseases is an area of growing interest. In the hypothalamus, restorative gene therapy has been successfully implemented in Brattleboro rats, an arginine vasopressin (AVP) mutant which suffers from diabetes insipidus, and in Koletsky (fa(k)/fa(k)) and in Zucker (fa/fa) rats which have leptin receptor mutations that render them obese, hyperphagic and hyperinsulinemic. In the above models, viral vectors expressing AVP, leptin receptor b and proopiomelanocortin, respectively, were stereotaxically injected in the relevant hypothalamic regions. In rats, aging brings about a progressive degeneration and loss of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons, which are involved in the tonic inhibitory control of prolactin secretion and lactotropic cell proliferation. Stereotaxic injection of an adenoviral vector expressing insulin-like growth factor I corrected their chronic hyperprolactinemia and restored TIDA neuron numbers. Spontaneous intermediate lobe pituitary tumors in a retinoblastoma (Rb) gene mutant mouse were corrected by injection of an adenoviral vector expressing the human Rb cDNA and experimental prolactinomas in rats were partially reduced by intrapituitary injection of an adenoviral vector expressing the HSV1-thymidine kinase suicide gene. These results suggest that further implementation of gene therapy strategies in neuroendocrine models may be highly rewarding.

DNA synthesis during hormone-dependent mammary tumor regression in rats.

A serial biopsy method was developed to study DNA synthesis in 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors during the regression process induced by bromocryptine (CB-154) administration in highly inbred female SD rats. With this technique the changes in tumor size could be correlated with those of DNA synthesis in single regressing tumors. DNA synthesis was estimated by the in vitro incorporation of tritiated thymidine into DNA which correlated well (correlation coefficient r = 0.95) with the in vivo mitotic activity of these neoplasms. Neither the biopsies themselves nor the estral status of the hosts affected significantly the rate of tumor DNA synthesis. DNA synthesis decreased sharply 4-8 days after the beginning of CB-154 treatment, whereas tumor shrinkage occurred more gradually. In conclusion, the serial biopsy method is a reliable technique for the estimation of changes in DNA synthesis in regressing DMBA-induced rat mammary tumors, whereas the measurement of the rate of tumor shrinkage is not.

Expression of transgenes in normal and neoplastic anterior pituitary cells using recombinant adenoviruses: long term expression, cell cycle dependency, and effects on hormone secretion.

Adenovirus vectors have recently been used to transfer genes into a variety of cell types, including neurons, glial cells, Schwann cells, and epithelial cells. To evaluate the efficiency of gene transfer into pituitary cells using viral vectors, we used replication-deficient recombinant adenovirus vectors (RAds) encoding beta-galactosidase driven by various viral promoters. We tested the ability of RAds to infect and express beta-galactosidase within the different identified cell populations of the anterior pituitary anterior pituitary gland and also in tumor cells of anterior pituitary origin, i.e. GH3 and AtT20 cells. Our results demonstrate that transgenes encoded by RAds are expressed within all cell types of the adenohypophysis in vitro and also within AtT20 and GH3 endocrine tumor cells. Our long term expression studies indicate that long term expression with low cytotoxicity can be achieved, but that the longevity of transgene expression from RAds depends on the proliferative status of the target cells. Slowly dividing cells (endocrine population) express transgenes for longer than actively dividing cells (tumor cells and nonendocrine anterior pituitary cells). The ability of anterior pituitary cells to secrete ACTH or LH through the regulated secretory pathway decreased after infection with RAds at high multiplicity of infection (> or = 20 plaque-forming units/target cell), whereas cell viability was not affected. We also demonstrate that a higher percentage of cells expressed the transgene beta-galactosidase when we infected actively dividing GH3 cells compared with the infection of growth-arrested GH3 cells. This could reflect differential virus entry or differential activity of the individual promoters during different stages of the cell cycle. This work demonstrates that high efficiency gene transfer into all pituitary cell types can be achieved with RAds, and that this system can be exploited to characterize and experimentally manipulate pituitary-specific gene expression. The higher efficiency of infection and transgene expression in actively dividing cells compared to that in their growth-arrested counterparts could also be exploited for the treatment of pituitary adenomas that do not respond to classical treatment strategies, using suicide or cytotoxic gene therapy.

Changes in somatotropin and thyrotropin secretory patterns in aging rats.

In order to clarify whether pituitary enlargement influences the secretory patterns of growth hormone (GH) and thyrotropin (TSH) in old rats, we studied the correlation between pituitary weight and plasma levels of GH and TSH in Sprague-Dawley rats of different age and sex. Young female (3-4 months; YF), old female (25 months; OF), and senescent female (33-35 months; SF) rats and young male (3-4 months; YM) and old male (24-26 months; OM) rats carrying chronic intraatrial cannulas were used. Sequential blood samples were removed through the cannulas while the animals remained conscious and undisturbed. Plasma TSH and GH as well as serum thyroxine (T4) and triiodothyronine (T3) were measured by radioimmunoassay. At two years of age, both males and females showed a consistent decline in GH pulse amplitude without change in trough levels. By 33-35 months of age, females showed a reversal in the previous pattern of change for GH secretion: pulse amplitude, trough levels, and mean plasma GH increased significantly with respect to the old females. The correlation between mean plasma GH and anterior pituitary (AP) weight was positive and significant (p less than 0.01) for females but nonsignificant for males. Old and senescent rats showed significantly lower serum T4, but not T3, than young animals while plasma TSH increased with age in both sexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Degradation of immunoreactive albumin in young and old conscious female rats.

The kinetics of inactivation of plasma albumin was studied in young (3-4 months) and old (25-28 months) Sprague-Dawley female rats. Conscious, free-moving animals carrying indwelling atrial and carotid cannulas received a single injection of [125I]-albumin (rat) via the carotid cannula. Sequential blood samples were removed at intervals during the following 120 min, and total (TR) and immunoprecipitable radioactivity (IPR) were determined in the corresponding plasmas. TR disappearance curves for young and old animals were almost identical but IPR disappearance curves showed a significantly faster decline in the young rats. The absolute plasma volumes for young and old rats were (mean +/- S.E.M.), 10.8 +/- 1.1 and 14.4 +/- 1.5 ml, respectively (P less than 0.05). The IPR/TR ratio, an estimate of albumin inactivation within the plasma space, showed a monoexponential decrease in vivo with a t 1/2 of 11.4 +/- 5.1 and 39.3 +/- 10.8 h (P less than 0.05) for young and old rats, respectively. The in vitro t 1/2s for albumin were 5.25 +/- 1.02 and 3.42 +/- 0.91 days (NS) for young and old rats, respectively. It is concluded that: the rate of albumin catabolism declines with age in the female rat; albumin is mainly inactivated in the extravascular space; and total plasma volume increases significantly with age in this species.

Reduced ability of hypothalamic and pituitary extracts from old mice to stimulate thymulin secretion in vitro.

There is substantial evidence that growth hormone (GH) is particularly important in the control of the age-related decline of thymus function. It was therefore of interest: (a) to assess the overall capacity of tissue extracts from mediobasal hypothalamus (MBH), anterior pituitary (AP) and testis, obtained from young (3 months, Yc), middle-aged (13 months, MAc) and old (18 months, Oc) intact C57BL/6 mice to stimulate in vitro the release of thymulin, a Zn-bound immunoregulatory thymic peptide, from pure cultures of mouse thymic epithelial cells (TEC); (b) to perform the same evaluation utilizing MBH, AP and testicular extracts from mice of the same age-range but treated for 45 days with a sc dose of ovine GH (2 micrograms/g body wt) known to stimulate thymulin secretion in vivo. Pituitary hormones were measured by heterologous rat RIAs, whereas thymulin release was estimated by a rosette assay. Untreated animals showed a significant age-dependent increase in the AP content of follicle stimulating hormone but not in other AP hormones. In both control and treated animals, pituitary GH content decreased significantly with age. MBH extracts from C57BL/6 males evidenced thymulin-releasing activity on mouse TEC lines. This activity was maximal in the MBH from young animals and declined with the age of the MBH donors. The thymulin-releasing activity of MBHs from GH-treated mice was higher than that of the control animals and showed a less pronounced decline with age. AP extracts from the same animals showed a higher thymulin-releasing activity than did MBH preparations. This activity showed a progressive age-associated reduction in the APs from untreated mice, whereas in the GH-treated group, an age-related decline was only seen in the old donors. Control testicular extracts had little effect on thymulin release whereas GH treatment induced a definite thymulin-release inhibiting activity in the testicular homogenates of our animals which increased progressively with the age of the testis donors. We conclude that the MBH, AP and testis of the young mouse contain factors able to affect directly the endocrine activity of the thymic epithelium. The amount of these substances declines with age and seems to be modulated by GH.

Thymulin stimulates prolactin and thyrotropin release in an age-related manner.

Thymulin is a Zn-bound nonapeptide produced by the thymic epithelial cells (TEC) whose secretion is modulated by prolactin (PRL) and thyroid hormones, among other hormones. We assessed the ability of thymulin to influence the release of PRL and thyroid stimulating hormone (TSH) from dispersed anterior pituitary (AP) cells from young, middle-aged and senescent Sprague-Dawley female rats. Perifused and incubated AP cells were used in different sets of experiments and PRL and TSH release was measured by radioimmunoassay. Perifusion of young and senescent AP cells with thymulin doses ranging from 10(-8) to 10(-5) M gave a logarithmic dose response pattern for both hormones. Supernatants from TEC lines also showed PRL and TSH secretagogue activity. Hormone release was always lower in the senescent cells. AP cells incubated with 10(-8) to 10(-3) M thymulin showed a time- and dose-dependent response for both hormones, the latter being bell-shaped with a maximum at 10(-7) M thymulin. Preincubation of thymulin with an anti-thymulin serum completely quenched the secretagogue activity of the thymic hormone. Coincubation of thymulin with TRH revealed a synergistic release of PRL and TSH in AP cells from all age groups. The calcium chelator EGTA but not the calcium ionophore A23187, blocked the hormone-releasing activity of thymulin in AP cells. The cAMP enhancers, caffeine, NaF and forskolin, significantly increased the thymulin-stimulated release of PRL and TSH, while trifluoperazine, a protein kinase C inhibitor, had no effect. The inositol phosphate enhancer LiCl, potentiated the action of thymulin on PRL and TSH. The present results suggest that the TRH-like activity documented here for thymulin is a receptor-mediated effect which appears to involve calcium, cAMP and inositol phosphates. Senescence but not middle age, appears to impair AP cell responsiveness to thymulin.

Immune-neuroendocrine interactions during aging: age-dependent thyrotropin-inhibiting activity of thymosin peptides.

Thymosin fraction 5 (TF-5), a partially purified thymic preparation, has been previously shown to have luteinizing hormone-releasing hormone (LH-RH)-releasing activity in perfused rat hypothalamus as well as in vivo stimulatory effect on the pituitary-adrenal axis in prepubertal monkeys. We report here the effect of TF-5 on the TSH-thyroid axis in young (3 months) and old (25 months) Sprague-Dawley male rats. Conscious free-moving animals carrying an indwelling atrial cannula received a single dose of 5 mg/kg body wt. of either bovine serum albumin (BSA) or TF-5 via the cannula. In the young rats, TF-5 induced a marked reduction of plasma thyrotropin (TSH) which was significantly greater than the normal circadian decline observed in the BSA-treated controls. The old males displayed high basal levels of TSH which showed no circadian rhythmicity, and did not respond to TF-5. Thyroxine (T4), triiodothyronine (T3), corticosterone, and prolactin levels were not affected by TF-5 at the dose levels tested. The old rats had significantly lower basal levels of T4, but not T3, than their young counterparts. The synthetic peptides thymosin alpha-1 and serum thymic factor, which are components of TF-5, had no effect on the above hormones when injected in doses up to 5 micrograms/kg body wt. Acute thymectomy in 3-month-old males induced a significant increase in basal levels of TSH without affecting plasma T4 or T3. These results suggest that the thymus has an inhibitory action on TSH in the rat, which is not mediated by the thyroid gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-dependent prolactin-releasing activity of nucleoproteins.

In previous studies we demonstrated that histone preparations possess multiple effects in vivo on pituitary hormone secretion and that these effects tend to disappear with age. We have now evaluated the in vitro effects of histone and nucleohistone preparations on the secretion of prolactin (PRL) in perifused pituitary cells from young (4 months) and senescent (29-33 months) female rats. Freshly dispersed pituitary cells were packed into short columns and were continuously perifused with serum-free medium. The substances to be tested were pumped through the perifusion circuit, at the end of which perifusate fractions were collected and hormones measured by specific radioimmunoassay (RIA). Quantitative immunohistochemistry was carried out on the pituitary glands from seven young and six senescent females. In vitro basal PRL release was similar in both age groups. Perfusion of cells with median eminence extract (1/90 to 1/10), histone H2A (100 to 1000 micrograms/ml) or nucleohistone (200 to 1000 micrograms/ml), generated PRL responses which were higher in young than in senescent cells. The pituitaries of the senescent animals were characterized, in most cases, by the presence of chromophobic microprolactinomas against a background of diffuse prolactotroph hyperplasia. Our results confirm previous evidence that circulating nucleohistones and histones may act as hypophysotropic signals. The morphologic alterations in PRL cell populations found in the sencscent rats may play role in the desensitization of the pituitary gland to nucleoproteins, and possibly to other hypophysiotropic molecules, with age.

Half-life of plasma growth hormone in young and old conscious female rats.

The kinetics of disappearance of plasma GH was studied in young (3-4 months) and old (24-27 months) Sprague-Dawley female rats. Conscious, free moving animals carrying indwelling atrial and carotid cannulas received a single injection of 125I-rGH via the carotid cannula. Sequential blood samples were removed at intervals during the following hour, and total (TR) and immunoprecipitable radioactivity (IPR) were determined in the corresponding plasmas. Both TR and IPR displayed biexponential kinetics in vivo which did not differ significantly, for each variable, between young and old animals. The volumes of distribution of GH were also similar in both age-groups. The IPR/TR ratio, an estimate of GH inactivation within the plasma space, showed a decreasing sigmoid-shaped kinetics in vivo with a time of semi-inactivation (ti1/2) of 23.8 +/- 1.2 and 29.0 +/- 1.0 min (mean +/- SE) for young and old rats, respectively (P less than 0.02). The estrous status did not significantly affect ti1/2 values in vivo. The in vitro t1/2 was estimated by incubating plasma from the young and old animals at 37 degrees C with 125I-rGH for several hours. The IPR/TR ratio displayed a linear kinetics in vitro with t1/2 values of 23.7 +/- 1.7 and 25.8 +/- 1.9 h (NS) for young and old animals, respectively. The above results show that GH catabolism decreases slightly with age in the female rat, although it is unlikely that this change has a significant effect on plasma levels of GH. The data also suggest that GH is physiologically inactivated in the extravascular space.

Use of recombinant herpes simplex virus type 1 vectors for gene transfer into tumour and normal anterior pituitary cells.

In this paper we demonstrate the use of recombinant viral vectors derived from herpes simplex virus type 1 (HSV1) to transfer reporter genes in vitro into rat anterior pituitary cells grown in primary cultures and the anterior pituitary tumour cell lines GH3 and AtT20. The three vectors used were, tsK/beta-galactosidase (beta-gal), tsK/CRH and tsK/TIMP, the corresponding transgene products respectively being E. coli beta-gal, pre-procorticotropin releasing hormone (ppCRH), and the chimeric protein TIMP/Thy1 (tissue inhibitor of metalloproteinases (TIMP)/linked to the carboxy terminus of Thy1 which confers the addition of a glycolipid glycosyl-phosphatidylinositol anchor in the ER). Double labelling immunofluorescence experiments to detect reporter proteins and transduced cell types indicated that the three vectors could transfer and express the reporter genes in normal and tumour anterior pituitary cells. Virus infection of pituitary cells was characterised, and it was shown that infection with tsK/beta-gal at multiplicities of infection (MOI)=10, 100% of tumour and non-endocrine anterior pituitary cells expressed beta-gal, whereas 75% endocrine anterior pituitary cells expressed the transgene. Long-term expression studies after infection with tsK/beta-gal indicated that anterior pituitary cells in primary cultures expressed the transgene for significant longer periods than tumour anterior pituitary cells. Growth arrest by serum starvation markedly decreased the frequency of transgene expression in anterior pituitary cells following infection with tsK/beta-gal. Transgenic products expressed from tsK were targeted to their correct intracellular domain in both anterior pituitary cells in primary cultures and in pituitary tumour cell lines. We conclude that transgenes can be delivered into anterior pituitary cells in primary culture and pituitary tumour cell lines using tsK derived HSV1 vectors. The prospect of employing viral vectors to transfer genes into endocrine cells opens up the potential exploration of various molecular aspects of pituitary cell function both in vitro and in vivo, as well as the use of gene transfer into the pituitary for potentially therapeutic applications, such as the treatment of pituitary tumours.

Studies on the gonadotropin-releasing activity of thymulin: changes with age.

We assessed the ability of thymulin, a zinc-dependent nonapeptide produced by the thymic epithelial cells, to influence the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from dispersed anterior pituitary (AP) cells from young, adult, and senescent female rats. Perifusion of young and senescent AP cells with thymulin doses of 10(-6) to 10(-5) M gave a significant stimulatory response for LH but not FSH. Gonadotropin release was always lower in the senescent cells. AP cells from both age groups incubated with 10(-8) to 10(-3) M thymulin showed a time- and dose-dependent response for both gonadotropins, with a maximal stimulation at 10(-7) M. Preincubation of thymulin with an antithymulin serum completely quenched the secretagogue activity of the hormone. Coincubation of thymulin with the secretagogue gonadotropin-releasing hormone (GnRH) revealed a synergistic effect on LH release and an additive effect on the release of FSH. The calcium chelator EGTA blocked the gonadotropin-releasing activity of thymulin in AP cells. The cAMP enhancers, caffeine, NaF, and forskolin significantly increased the thymulin-stimulated release of gonadotropins. The inositol phosphate enhancer LiCl potentiated the action of thymulin on gonadotropins. It is concluded that the gonadotropin-releasing activity documented here for thymulin is an age- and receptor-dependent effect mediated in part by calcium, cAMP, and inositol phosphates.

Thyrotropin-releasing activity of histone H2A, H2B and peptide MB35.

Histones possess multiple hormone-like activities. We studied the specificity and signal transduction pathways involved in the thyrotrophin (TSH)-releasing activity of histones H2A, H2B and peptide MB35, a H2A fragment, using perifused and incubated dispersed rat pituitary cells and measuring TSH release by a specific R1A. Histones released TSH in a dose- and time-dependent fashion while peptide MB35 behaved as a weaker secretagogue. Trifluoperazine and EGTA blocked histone-stimulated TSH release while forskolin and other cAMP enhancers did not. We conclude that the TSH-releasing activity of histones H2A and H2B is mediated by calcium- and diacylglycerol-associated pathways.

Histones and related preparations interfere with immunoassays for peptide hormones.

We report here that histones and certain related preparations generate a consistent interference with radioimmuno (RIA), immunoradiometric (IRMA), and enzyme-linked immunosorbent (ELISA) assays for a number of peptide hormones. Histones H1, H2A, H2B, H3, HIIA, HIIS, protamine, and the related preparations homeostatic thymus hormone and peptide MB35 generated a dose-dependent signal in both the human corticotropin-releasing hormone (CRH) and the human adrenocorticotropic hormone (ACTH) IRMA. This signal was not affected when the linker antiserum was removed from the IRMA reagent mixture, thus proving that the signal was not due to cross-reaction or sample contamination with CRH or ACTH. The above histone preparations, as well as protamine, but not ubiquitin, also generated a strong negative interference with RIAs for ACTH, CRH, rat growth hormone (rGH), and rat prolactin (rPRL). In an ELISA system for the thymic peptide facteur thymique sérique, histones and protamine again showed a strong interfering activity. When known amounts of rGH, rPRL, and hACTH were dissolved in charcoal-washed horse serum or supernatants from rat liver homogenates (centrifuged 1 h at 10,000 x g), and the corresponding RIAs and IRMA (for ACTH) were performed in the absence or presence of histones HIIA and HIIS (at 1 mg/ml level), an interfering activity of histones was again observed. We conclude that histones and some related peptide preparations have, when present in biologic fluids, a significant capacity to interfere with peptide immunoassays.

Studies on the prolactin-releasing mechanism of histones H2A and H2B.

In previous studies we demonstrated that histone preparations possess multiple effects in vivo on pituitary hormone secretion. We have now studied the specificity and signal transduction pathways involved in the prolactin (PRL)-releasing activity of histones H2A and H2B on perifused and incubated rat pituitary cells. In the perifusion experiments, freshly dispersed pituitary cells were packed into short columns and were continuously perifused with serum-free medium. The substances to be tested (stimuli) were pumped through the perifusion circuit, at the end of which perifusate fractions were collected and PRL measured by specific RIA. In the incubation studies, freshly dispersed pituitary cells were incubated in a metabolic incubator with different stimuli at different doses and for varying times. Perifusion of cells with median eminence extract (1/30), histone H2A (30 microM) or histone H2B (30 microM), generated clear PRL release responses. Cells incubated with histone H2A and H2B showed a dose- and time-dependent stimulatory effect on PRL release which, for H2A, was blocked by peptide MB-35, an 86-120 amino acid synthetic fragment of histone H2A. The polycation, poly-lys was unable to mimic the action of histones. To detect the possible signal transduction pathways involved in the response of lactotrophs to histones, cells were incubated with the calcium ionophore A23187, the calcium chelator EGTA, the intracellular phosphoinositide enhancer LiCl, the intracellular cAMP enhancers caffeine, NaF and forskolin, and the protein kinase C inhibitor, trifluoperazine (TFP). Both EGTA (or EGTA plus A23187 ionophore) and TFP were able to reduce significantly the response of lactotrophs to histones. Our results confirm previous evidence that histones may act as hypophysotropic signals. The data also suggest that calcium- and diacylglycerol-associated pathways participate in these effects.

Corticotropin-releasing (CRH) activity of thymic peptides on CRH-insensitive corticotropic tumor cells.

A number of thymic preparations are known to stimulate corticotropin (ACTH) release from pituitary cells but it remains unclear whether this effect is mediated by the corticotropin-releasing hormone (CRH) receptor-associated pathway. We report here that thymosin fraction five (TF5), peptide MB-35 and possibly calf thymus histones can stimulate the release of ACTH from a CRH-insensitive variant of the mouse corticotropic cell line AtT20. The effective concentration range at which TF5 and MB-35 displayed their ACTH-releasing activity in a dose-dependent manner was 100 to 2,000 micrograms/ml and 10 to 100 ng/ml, respectively, whereas neither preparation induced a significant depletion of intracellular ACTH stores. Our data suggest that thymosin peptides can stimulate ACTH release from corticotrophs by a CRH receptor-independent mechanism.

Glial cell line-derived neurotrophic factor gene therapy ameliorates chronic hyperprolactinemia in senile rats.

Progressive dysfunction of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons during normal aging is associated in the female rat with chronic hyperprolactinemia. We assessed the effectiveness of glial cell line-derived neurotrophic factor (GDNF) gene therapy to restore TIDA neuron function in senile female rats and reverse their chronic hyperprolactinemia. Young (2.5 months) and senile (29 months) rats received a bilateral intrahypothalamic injection (10(10) pfu) of either an adenoviral vector expressing the gene for beta-galactosidase; (Y-betagal and S-betagal, respectively) or a vector expressing rat GDNF (Y-GDNF and S-GDNF, respectively). Transgenic GDNF levels in supernatants of GDNF adenovector-transduced N2a neuronal cell cultures were 25+/-4 ng/ml, as determined by bioassay. In the rats, serum prolactin (PRL) was measured at regular intervals. On day 17 animals were sacrificed and neuronal nuclear antigen (NeuN) and tyrosine hydroxylase (TH) immunoreactive cells counted in the arcuate-periventricular hypothalamic region. The S-GDNF but not the S-betagal rats, showed a significant reduction in body weight. The chronic hyperprolactinemia of the senile females was significantly ameliorated in the S-GDNF rats (P<0.05) but not in the S-betagal rats. Neither age nor GDNF induced significant changes in the number of NeuN and TH neurons. We conclude that transgenic GDNF ameliorates chronic hyperprolactinemia in aging female rats, probably by restoring TIDA neuron function.

Changes in chromatin composition associated with hormone-dependent mammary tumor regression.

The chemical composition of chromatin from dimethylbenz(a)anthracene (DMBA)-induced mammary tumors was compared in growing and regressing neoplasms. Tumor regression was induced by 2-bromo-alpha-ergocryptine (CB-154) administration to the tumor-bearing rats. Seventy percent of the neoplasms showed a sharp fall in the levels of 3H-thymidine incorporation into DNA after 7-8 days of treatment (1 mg CB-154/day). These CB-154-responsive tumors showed a significant increase in nonhistone chromosomal proteins (NHCP) relative to DNA, while the histone fraction remained constant. Non-responsive tumors showed no change in their chromatin composition. Low doses of ovine prolactin (18 IU) given together with CB-154 (1 mg) were able to prevent the effects of the latter on both DNA synthesis and chromatin composition of DMBA- induced mammary tumors. High doses of hormone (40 IU) produced a two-fold increase in the tumors' DNA synthesis irrespective of the simultaneous administration of 1- or 2-mg doses of CB-154. This high dose of hormone was also able to prevent the effects of CB-154 on chromatin composition of the tumors. We conclude that the above effects of CB-154 are exerted mainly, if not exclusively, through the inhibition of prolactin secretion. On the other hand, the regression of these neoplasms after prolactin deprivation seems to be associated with an increase in the NHCP/DNA ratio. The possibility of an involvement of tumor cell membrane in the mechanism of regression of prolactin-dependent mammary tumors is discussed.

Diminished diurnal secretion of corticosterone in aging female but not male rats.

Since very little is known about the impact of aging on adrenocortical function in female rats, it was of interest to compare the age changes in the circulating levels of adrenocorticotropin (ACTH) and corticosterone in male and female Sprague-Dawley rats, and correlate these changes with those in plasma prolactin (Prl), which is known to stimulate adrenal steroid secretion. Hormones were measured in young (3-4 months) and old (24-26 months) male rats as well as in young (3-4 months), old (25 months) and senescent (33-35 months) female rats. Sequential plasma samples were obtained from chronically cannulated animals every 30 min from 11:30 a.m. to 4:30 p.m. ACTH was measured in trunk serum--obtained between 11:30 a.m. and 1:30 p.m.--whereas corticosterone and prolactin were measured in plasma using specific RIAs for the corresponding hormones. No age changes were detected in serum ACTH in either sex. The integrated values of plasma corticosterone did not change with age in males but decreased significantly (p less than 0.05) in old and senescent as compared to young females. Plasma corticosterone was higher in young females than in young males but this sex-related difference disappeared with age. Plasma Prl increased significantly with age in both male (p less than 0.05) and female (p less than 0.001) rats but showed no significant correlation with corticosterone levels. The present results suggest that ACTH secretion does not show major age- or sex-related changes. Our corticosterone data are compatible with the idea that gonadal aging has a significant impact on plasma glucocorticoid regulation in female rats but not in males.