Tolerance of 5-fluorodeoxyuridine resistant human thymidylate synthases to alterations in active site residues.

Fluoropyrimidines, such as 5-fluorouracil (5-FU), are used extensively in cancer therapy. In the cell, 5-FU is metabolized to 5-fluorodeoxyuridylate (5-FdUMP), a tight binding covalent inhibitor of thymidylate synthase (TS). In order to create 5-FdUMP resistant enzymes to protect chemosensitive normal cells and further understand mechanisms of 5-FdUMP resistance, we have randomized four residues within the active site of TS. Our previous studies identified alterations in residues which produce active TS with enhanced resistance to 5-fluorouridine (5-FdUR). By remutagenizing a subset of the 13 previously targeted residues (A197, L198, C199 and V204), an unbiased random library can be created allowing for extensive testing of all possible amino acid substitutions at each of the sites. Using genetic complementation and selection in Escherichia coli, we identified the spectrum of substitutions that yield active TS as well as those that resulted in 5-FdUR resistant mutants of TS. The 5-FdUR resistant TS were found to share several structural features including hydrophobic substitutions at residue 197, retention of the wild-type leucine 198, the alteration C199L (present in 64% of the drug-resistant library), and polar alterations of valine 204. The catalytic activity of mutants with these features was approximately equal to that of the wild-type TS.

The ontogeny of sex differences in estrogen-induced progesterone receptors in rat brain.

The sex and age-related changes in the concentration of cytosol progesterone receptors (CPR) induced by estrogen (E) treatment in rat brain and pituitary were investigated by a modification of the Palkovits punch procedure using fresh tissue. Young male and female gonadectomized rats, 15, 21, 30, and 42 days of age, were treated for 44 h by a single sc injection of synthetic E. [Moxestrol (Ru2858)]. Adult gonadectomized animals were treated for 72 h by three injections of estradiol benzoate. Cytosol from pituitary and from punches of ventromedial nuclei (VMN), medial preoptic area (mPOA), arcuate nucleus (ARC), and cerebral cortex was labeled with 0.4 nM [3H]promegestrone (Ru5020) to maximize signal to noise and specificity of labeling of CPR. The developmental patterns of CPR differed across brain regions and between males and females. In VMN, females showed higher CPR levels after estrogen priming at 15, 21, and 42 days of age and in the adult; in ARC, females showed higher CPR levels after E priming at 15, 21, and 30 days of age, but not at 42 days or in the adult. In mPOA and pituitary, no consistent sex differences in CPR induction were found. Cortex showed no induction of CPR by E priming. Radioautography of [3H]Ru5020 uptake in VMN of E-primed 15-day-old male and female rats revealed significantly higher labeling in females, thus showing that the CPR levels in vitro reflect a difference in hormone retention in vivo. Female VMN contained more neurons with a higher labeling index than male VMN. Before puberty (approximately = 30 days), there was a decline in CPR levels induced by E priming in both sexes in pituitary, ARC, mPOA, and VMN. At 30 days, the female greater than male sex difference disappeared and tended to reverse in mPOA and VMN, only to be restored again by 42 days of age. Thus, the CPR induction by E priming may reflect underlying changes in E-sensitive brain regions associated with the preparation for puberty, as well as underlying sex differences in response to estrogen programmed by perinatal exposure to testosterone.

Autoradiographic localization of estradiol-binding neurons in the rat hippocampal formation and entorhinal cortex.

This study has examined the distribution of [3H]estradiol and [1 alpha,2 alpha-3H]testosterone uptake in the hippocampal formation and entorhinal cortex of male and female rats. In both males and females, [3H]estradiol-binding neurons in Ammon's horn are located deep in stratum pyramidale and may correspond either to polymorphic interneurons or to early maturing pyramidal cells. Interneurons of strata oriens, lucidum and radiatum of Ammon's horn and of stratum moleculare of the subiculum also bind [3H]estradiol, as do basket cell interneurons in the polymorphic, infragranular layer of the dentate gyrus. While no granule cells appear to accumulate [3H]estradiol, these cells may be affected transsynaptically by gonadal steroids via their afferent contacts with the entorhinal cortex, which, of the areas examined, contains the greatest number of [3H]estradiol-binding neurons. While relatively few neurons concentrate [3H]estradiol in the hippocampal formation, these are localized to specific subpopulations, which may enhance their functional significance. Because there is no significant nuclear accumulation of [3H]-alpha-testosterone in either the entorhinal cortex or hippocampal formation, it appears that aromatase enzyme activity is not a major contributor to estrogen receptor occupancy in adult rats.

Biochemical and radioautographic analysis of estrogen-inducible progestin receptors in female ferret brain and pituitary: correlations with effects of progesterone on sexual behavior and gonadotropin-releasing hormone-stimulated secretion of luteinizing hormone.

Cytosolic binding molecules for the synthetic progestin [3H]R5020, were isolated in vitro from several brain regions including preoptic area/anterior hypothalamus, mediobasal hypothalamus, medial amygdala and parietal cortex as well as the pituitary gland of ovohysterectomized female ferrets. Binding of [3H]R5020 to cortical cytosols was saturable, of high affinity (apparent dissociation constant of 2.0 nM), and was steroid specific. Pretreatment of ferrets with a Silastic capsule containing estradiol caused significant increments in the concentration of cytosolic R5020 binding sites in hypothalamus and pituitary gland, but not in the other brain regions studied. Brains of additional ovohysterectomized ferrets, which had been primed with estradiol prior to receiving an i.v. injection of [3H]R5020, were processed radioautographically. Intense labeling of cells was seen in the medial and lateral preoptic area, in the lateral hypothalamus, and in the ventromedial and arcuate nuclei of the hypothalamus. Radioautograms from the brains of additional ovohysterectomized ferrets given an i.v. injection of [3H]estradiol revealed labeled cells in all of the above regions, in addition to the basolateral portion of the septum, the bed nucleus of the stria terminalis, and in the anterior amygdaloid area as well as the medial and cortical nuclei of the amygdala. This distribution of neural progestin and estrogen binding sites resembles those previously reported for these steroids in the rat, guinea pig, hamster and macaque. Functional studies showed that acute s.c. implantation of a Silastic capsule containing progesterone to ovohysterectomized ferrets, which had been primed with a low dosage of estradiol, failed to augment their sexual receptivity in limited tests with stimulus males given 4 and 8 h after progesterone treatment. This result contrasts with the well-established facilitatory effect of progesterone on sexual receptivity in rat and guinea pig. Chronic exposure to a progesterone capsule caused significant reductions in sexual receptivity in ovohysterectomized ferrets which were implanted concurrently with a second Silastic capsule containing a high dosage of estradiol. Similar effects of progesterone have been reported in rat and guinea pig, but not in the rhesus monkey. Thus species differences in the ability of progesterone to facilitate or inhibit sexual receptivity are not readily explained by species differences in the neural distribution of estrogen-induced progestin receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Estrogen and progestin receptors appear in transplanted fetal hypothalamus-preoptic area independently of the steroid environment.

Sexual maturation and differentiation of the rat brain are believed to result from the interaction of gonadal steroids with specific neural receptors during late fetal and early postnatal life. A variety of evidence indicates that the first appearance of estrogen receptors in the hypothalamus-preoptic area (HPOA) during the perinatal period is a crucial evident underlying these processes. However, it is unknown to what extent the ontogeny of estrogen receptors is itself influenced by gonadal steroids present in the fetal environment. In order to address this question, estrogen receptors were assayed in HPOA 8 weeks after transplantation of the tissue from embryonic day 15 to 18 fetuses to either the choroidal pia overlying the superior colliculus or to the anterior chamber of the eye of adult female hosts. Host animals were either intact or ovariectomized and adrenalectomized, with or without estrogen replacement. The saturable binding of estradiol to cytosol of HPOA transplants exhibited the steroid specificity and high affinity characteristic of authentic estrogen receptors. No differences in the level of cytosol estrogen receptors in transplanted HPOA grown in the presence or absence of gonadal steroids were found. Receptor concentrations were also similar in HPOA taken from male or female fetuses. Autoradiography with [3H]estradiol revealed clusters of estrophilic cells in the transplants similar to those of the adult host hypothalamus, again regardless of whether the transplant developed in the presence of gonadal steroids. Estrogen receptors from both groups were also found to be biochemically functional as indicated by the ability of acute estrogen treatment to induce progestin receptors in the transplants.(ABSTRACT TRUNCATED AT 250 WORDS)

Perinatal development of hypothalamic and cortical estrogen receptors in mouse brain: methodological aspects.

Binding of the estrogen, [3H]moxestrol, to fetal and neonatal mouse brain cytosol receptors was examined to determine the ontogeny of estrogen receptors in the hypothalamus and cerebral cortex from embryonic (E) day 15 to postnatal (P) day 18. Cytosol receptor assays were performed under exchange conditions at 25 degrees C for 4 h in order to measure receptors which had become occupied by estradiol during tissue homogenization. Scatchard analysis revealed high affinity (Kd = 0.4 nM) sites and was in good agreement with single point assays at 1 nM, which measured 70% of binding capacity. Binding was initially examined in the whole forebrain. Total binding of [3H]moxestrol in the forebrain increases between E15 and E18 and reaches adult levels at P9. The increase in binding relative to protein content peaks at P9 and then decreases, whereas the amount of binding relative to DNA content reaches a maximum between P12 and P15. The developmental time-course of the estrogen receptors was studied in the hypothalamus and 3 cortical regions. In the hypothalamus binding of [3H]moxestrol increases from P5 to P18 of the cortical areas. The cingulate cortex shows the highest amount of binding, increasing until P9 and then declining. In the other two cortical areas studied, the lateral and posterior cortex, binding expressed per mg DNA, is somewhat higher between P7 and P15 than in adults. When the binding is expressed per mg protein there is a sharp decline after P7, the magnitude of which is probably a result of a large increase in protein content relative to amount of receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Perinatal development of estrogen receptors in mouse brain assessed by radioautography, nuclear isolation and receptor assay.

The development of estrogen receptors was investigated in vivo in the brains of fetal and neonatal mice 2 h after administering [3H]moxestrol to the pregnant mothers or neonates. Moxestrol bypasses the alpha-fetoprotein 'protective barrier' and gains access to estrogen receptors. Analysis of [3H]moxestrol uptake by radioautography and by cell nuclear isolation and counting of radioactivity revealed a marked increase in the number of estrogen receptors and estrophilic cells in the brain during late fetal and early postnatal development. Assays of cytosol estrogen receptors were conducted in parallel and revealed a comparable pattern of development. The increase in estrogen receptors and labeling was especially great from embryonic day (E) 15 to E18. Cytosol assays revealed a low level of receptors in the whole brain on E13. Radioautography revealed that clearly labeled cells in the hypothalamus and preoptic area were virtually absent on E13 but were evident on E15, with marked increases occurring between E15 and E18, both in number of labeled cells and in intensity of labeling per cell. Within the cerebral cortex the dorsal cingulate cortex was the most extensively labeled area; however, clearcut labeling was not evident on E13 or E15. Thus, the development of cortical estrogen receptors occurs somewhat later than that in the hypothalamus and preoptic area. The perinatal increase in estrogen receptors usually begins several days after the birthdates of neurons in these estrophilic regions of the brain, and corresponds to the early responsiveness of these neurons to the organizational and activational influences of estrogen.

Evidence for glucocorticoid target cells in the rat optic nerve. Hormone binding and glycerolphosphate dehydrogenase induction.

Biochemical evidence suggests that neuroglia are responsive to glucocorticoids, yet previous studies of glucocorticoid localization have typically failed to demonstrate significant uptake by neuroglial cells. To further investigate this problem, we measured glycerol-3-phosphate dehydrogenase (GPDH) activity and glucocorticoid receptor binding capacity in normal rat optic nerves and in those undergoing Wallerian (axonal) degeneration. Binding studies were also performed on hippocampus and anterior pituitary for comparison purposes. Normal optic nerve preparations possessed a high level of GPDH activity that was glucocorticoid-inducible and that increased further following axonal degeneration. Antibody inactivation experiments demonstrated the presence of more enzyme molecules in the degenerating nerve preparations. correlative immunocytochemical studies found GPDH-positive reaction product only in morphologically identified oligodendrocytes, a result that is consistent with the previously reported localization of this enzyme in rat brain. Optic nerve cytosol fractions displayed substantial high-affinity binding of both dexamethasone (DEX) and corticosterone (CORT) that, like GPDH, was elevated approximately two fold in degenerating nerves. Finally, in vivo accumulation of [3H]DEX and [3H]CORT by optic nerve and other myelinated tracts was examined using nuclear isolation and autoradiographic methods. Although neither steroid was found to be heavily concentrated by these tissues in vivo, a small preference for DEX was observed in the nuclear uptake experiments. These results are discussed in terms of the hypothesis that glial cells are targets for glucocorticoid hormones.

Differentiation of embryonic hypothalamic transplants cultured on the choroidal pia in brains of adult rats.

Hypothalmic tissue from 16 to 18-day fetal rats was transplanted onto the choridal pia overlying the superior colliculus in adult female rats. After survival periods of 2 weeks to 19 months, brains containing transplants were processed for monoamine fluorescence histochemistry, immunohistochemistry for three neuropeptides (LHRH, somatostatin, neurophysin), or for autoradiography in ovariectomized hosts that received [3 H] estradiol. Most of the transplants survived and retained or increased in size; 14 of 25 transplants examined by fluorescence histochemistry were found to contain median eminence-like structures. In almost all of the transplants that were stained for neuropeptides, beaded processes and occasional cell bodies were observed. Although immunoreactive fibers were found near blood vessels, no palisade arrangement typical of the normal median eminence was evident. Each of the hypothalamic transplants on which steroid autoradiography was performed contained clusters of estrophilic neurons, the intensity of labeling of which was comparable to that seen in the host hypothalamus. These results indicate that many characteristic morphological and chemical features of the hypothalamus, which are not evident in the 16 to 18-day fetus, are elaborated in transplants during the survival period in the host. Transplantation of fetal hypothalamus to adult choridal pia thus appears to be a valuable approach for studying the factors, humoral or neural, that regulate the differentiation of this brain region.

Autoradiographic localization of hormone-concentrating cells in the brain of the female rhesus monkey.

With autoradiographic procedures, cells which bind 3H-estradiol were found in preoptic, hypothalamic and limbic structures in the brains of ovariectomized, adult female rhesus monkeys. Estrogen-binding cells were seen in the medial preoptic area, medial anterior hypothalamus, ventromedial nucleus, and especially heavy labelling was seen throughout the extent of the arcuate (infundibular) nucleus of the hypothalamus. In limbic structures, cells in the bed nucleus of the stria terminalis and in the medial nucleus of the amygdala were well labelled. Systematic charting also revealed smaller numbers of estrogen-concentrating cells in other specific hypothalamic and limbic locations. In the anterior pituitary, significant numbers of basophils and acidophils were found to bind estrogen. Pars intermedia and the posterior lobe were virtually unlabelled. In the uterus, heavily labelled cells were seen in the endometrial stroma and in the myometrium. These autoradiographic findings agree with results of parallel biochemical experiments. In monkeys injected with 3H-corticosterone, the most extensive high-intensity binding found with autoradiography was in the hippocampus. Both pyramidal neurons and dentate gyrus granule cells were labelled. Biochemical experiments, also, showed highest cell nuclear accumulation of corticosterone in the hippocampus. Findings with estradiol in the rhesus monkey extend to primates conclusions based on autoradiographic experiments with steroid sex hormones in wide variety of vertebrates, including fish, amphibians, birds, and various mammalian species (Morell st al., '75a). All of these vertebrate forms have sex hormone-concentrating neurons, which are found in specific preoptic, hypothalamic and limbic structures. In the species studied, such hormone-concentrating neurons appear to be involved in the hormonal control of behavioral and pituitary function.

Rat brain binds adrenal steroid hormone: radioautography of hippocampus with corticosterone.

Tritiated corticosterone injected subcutaneously into adrenalectomized male rats 1 hour before killing produced intense labeling of the hippocampus in radioautograms prepared by a method that reduced or prevented diffusion of the radioactive material. The pyramidal neurons of the cornu ammonis and the granule neurons of the gyrus dentatus contained more radioactivity than did other regions of the brain; however, the intensity of labeling varied among adjacent neurons. The nuclei of many neurons were clearly labeled but radioactivity was relatively sparse in the cytoplasm, in the axons where they branch from cell bodies, and in adjacent neuropil.