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C B Lazier

Publications and source records attributed to C B Lazier.

At least 37 records · Page 2Linked to original sources
PubMed1990

Comparison of the effects of tamoxifen and of a tamoxifen analogue that does not bind the estrogen receptor on serum lipid profiles in the cockerel.

Administration of the nonsteroidal antiestrogen tamoxifen to cockerels results in dose- and time-dependent decreases in the levels of free and esterified cholesterol, phospholipids, and triglycerides in serum and in very low density and low density lipoprotein fractions. Similar changes can be elicited using a tamoxifen analogue, N,N-diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine.HCl (DPPE). Like tamoxifen, this compound is capable of binding antiestrogen binding sites and exhibits a relative binding affinity of 90% compared with tamoxifen (Ki approximately 4-5 nM). Unlike tamoxifen, DPPE shows no measureable affinity for the cockerel liver nuclear estrogen receptor. Further, DPPE exhibits no estrogen agonist or antagonist activity as measured at the level of synthesis of apolipoprotein II of very low density lipoprotein by liver, synthesis of ovalbumin by oviduct, or growth of the oviduct. Although it is possible that the lipid-lowering effects of tamoxifen result from the opposition of endogenous estrogen action in the cockerel, the similarity of the effects of tamoxifen and DPPE on the lipid profiles suggests common mechanisms that do not involve the estrogen receptor.

Animals↗
PubMed1990

Expression of endogenous and transfected apolipoprotein II and vitellogenin II genes in an estrogen responsive chicken liver cell line.

A recently described chicken liver cell line, LMH, was characterized to evaluate responsiveness to estrogen. Expression of the endogenous apolipoprotein (apo) II gene was induced by 17 beta-estradiol when LMH cells were cultured with chicken serum. The response was low and yielded apoll mRNA at only 0.3% of the level seen in estrogenized rooster liver. Higher levels of apoll mRNA were achieved when LMH cells were transiently transfected with an expression plasmid for estrogen receptor. A transfected apoll gene was strongly expressed only when cotransfected with receptor. Expression of the endogenous vitellogenin (VTG) II gene was not detected. However, when cotransfected with a receptor expression plasmid, VTG II reporter plasmids were expressed in LMH cells in response to 17 beta-estradiol. These results suggest that estrogen responsiveness of LMH cells is limited by the availability of functional receptor. Low levels of estrogen receptor mRNA were detected in LMH cells, and receptor binding sites and mRNA were greatly increased following transient transfection with a receptor expression plasmid. Using this transient transfection protocol, several VTG II reporter plasmids were compared in LMH cells and chick embryo fibroblasts. A plasmid containing VTG II estrogen response elements linked to a heterologous promoter was regulated by estrogen in both cell types. In contrast, reporter plasmids containing the VTG II promoter were regulated by estrogen in LMH cells but were not expressed at all in chick embryo fibroblasts. These results suggest that regulation of the VTG II gene involves cell type-specific elements in addition to estrogen response elements.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Antiestrogen binding sites: general and comparative properties.

Nonsteroidal antiesterogens, such as tamoxifen, bind estrogen receptors with relatively high affinity. They also bind with high affinity and distinctive specificity to another class of intracellular sites, often termed "antiestrogen binding sites" or "AEBS". These sites do not bind estrogens and their function is unknown. In this article we compare and contrast the binding specificity, intracellular localization, concentration, tissue distribution and detergent solubilization characteristics of AEBS from a number of species. In general the sites are found in highest concentration in microsomal fractions from liver. AEBS from chicken liver can be solubilized with CHAPS detergent giving rise to a detergent-associated complex with an apparent molecular mass of about 265 kDa.

Animals↗
PubMed1987

Tamoxifen-induced modification of serum lipoprotein phospholipids in the cockerel.

The administration of tamoxifen (Tam), a nonsteroidal antiestrogen, or of a diphenylmethane derivative of Tam that does not bind to the estrogen receptor (DPPE) of cockerels results in a marked decrease in the concentration of serum lipoprotein constituents with an apparent alteration in phospholipid composition. To establish the nature of changes in phospholipids, the molecular species of phosphatidylcholine (PC) and sphingomyelin (Sph) were isolated and characterized. Between 9 and 18 hr following the administration of Tam or DPPE, there was a marked decrease in the proportion of molecular species of serum PC containing C16 and C18 fatty acids, but there was an increase in the proportion of molecular species containing C20 and C22 polyunsaturated fatty acids. Fatty acid analyses revealed that this change was due to an increase in arachidonic and docosahexaenoic acids at the expense of oleic and linoleic acids. These proportional changes were due to an absolute decrease in serum of PC molecular species containing palmitic and stearic acids in association with oleic and linoleic acids with very little change in the absolute concentration of molecular species containing arachidonic and docosahexaenoic acids. By contrast, the composition of Sph, which contained palmitic acid as the major fatty acid, was not altered during treatment. It is concluded that the short-term effect of Tam and DPPE on plasma phospholipids of the cockerel is due to a selective conservation of PC containing long chain polyunsaturated fatty acids.

Animals↗
PubMed1987

Interactions of tamoxifen in the chicken.

The triphenylethylene antiestrogens are very potent antagonists of estrogen action in the chicken and manifest little agonist activity compared to their action in other species. The estrogen antagonism is most probably mediated by the estrogen receptor, to which tamoxifen binds with a Ki of 2.6 nM. Tamoxifen is readily metabolized by liver to 4-hydroxytamoxifen, which binds the liver nuclear estrogen receptor with a Ki of 0.1 nM. The Kd of the receptor is 0.7 nM. Estrogen receptor concentrations in liver from immature chickens are relatively low both in nuclear and cytosol fractions. Treatment with estradiol results in 10-fold up-regulation of the nuclear levels to give a total receptor concentration of about 2 pmol/g tissue. Tamoxifen can promote this up-regulation to a limited extent, but interpretation of experimental results is compromised by difficulties with exchange assays in the face of the very high binding affinity of 4-hydroxytamoxifen. Tamoxifen also binds with high affinity (Kd 2-4 nM) and distinctive specificity to antiestrogen binding sites (AEBS) present in a wide variety of chicken tissues and in the highest concentration in the liver (800 pmol/g tissue). Liver and serum contain ether-soluble components which can compete for binding of [3H]tamoxifen to the AEBS. The serum AEBS inhibitory activity is chromatographically heterogeneous and is associated with a sterol-like fraction as well as with a fatty-acid-containing fraction. Tamoxifen treatment of cockerels results in dose- and time-dependent decreases in serum free and esterified cholesterol, and in phospholipids and triglycerides. These changes may reflect estrogen-receptor-independent interactions of tamoxifen.

Animals↗
PubMed1986

Stimulation of estrogen receptor accumulation by estradiol in primary cultures of salmon hepatocytes.

Hepatocytes of Salmo salar in primary culture form confluent monolayers and can be maintained at 11 degrees centigrade in serum-free medium for 8 days with minimal cell loss. Cultured hepatocytes from immature male salmon contain estrogen receptor both in nuclear and cytosol fractions (2000 and 2400 cites/cell, respectively). A single addition of estradiol results in an increase in the nuclear receptor to a level of 23 000 sites/cell after 24 h. This nuclear receptor concentration is similar to that in liver of estrogen-treated salmon in vivo, and is much higher than has been found for any other egg-laying vertebrate. The cultured salmon hepatocytes thus represent a highly sensitive system for the study of estrogen receptor dynamics and vitellogenesis in vitro.

Animals↗
PubMed1985

Binding of oxygenated cholesterol metabolites to antiestrogen binding sites from chicken liver.

High affinity (KD 2-4 nM) binding sites (AEBS) for the triphenylethylene antiestrogen, tamoxifen, are found in chicken liver cytosol preparations. These sites exhibit affinity for triphenylethylene derivatives typical of those reported for AEBS in other systems. Although steroids and cholesterol do not compete for the sites, certain oxygenated cholesterol metabolites, particularly 7-ketocholesterol, do so. Hot ethanol extracts of chicken and human serum contain AEBS inhibitory activity. This activity is chromatographically heterogeneous, but a sterol-containing fraction has been isolated in which the 7-ketocholesterol concentration can account for the AEBS inhibitory activity of the fraction.

Animals↗
PubMed1985

Hepatic estrogen receptors and plasma estrogen-binding activity in the Atlantic salmon.

Livers of male and female immature Atlantic Salmon (Salmo salar) contain specific high-affinity [3H]estradiol binding sites in cytosol (Kd 2-4 nM, concentration about 0.6 pmol/g liver). Low levels of high-affinity binding are detectable in salt extracts of nuclei of untreated fish, but injections of estradiol result in transient depletion of the cytosol binder and in accumulation of high levels of binding sites in nuclear salt extracts (Kd 5-6 nM; concentration about 6 pmol/g liver). Both the cytosol and nuclear binding sites are temperature sensitive and are optimally assayed by incubation at 2 degrees. Both are specific for estradiol and diethylstilbestrol (DES) and no significant competition by dihydrotestosterone (DHT), progesterone, or hydrocortisone is seen. The triphenylethylene nonsteroidal antiestrogen, 4-hydroxytamoxifen, exhibits an affinity comparable to that of estradiol. The nuclear binding activity sediments with a coefficient of 3.6 S in salt-containing sucrose density gradients, and is stable on storage at -20 degrees for several months. The cytosol binder on the other hand is not stable on sucrose density gradients or on prolonged storage. Salmon plasma contains two [3H]estradiol binding components, one with a relatively high affinity for [3H]estradiol (kd 13 nM) and the other having a much lower affinity but present in high concentrations. The high-affinity plasma binder exhibits distinctive specificity with no affinity for DES or 4-hydroxytamoxifen but some affinity for DHT and progesterone. These properties serve to distinguish the plasma activity from the intrahepatic estrogen binders. The salmon liver estrogen receptor system has many features in common with typical estradiol receptors from other vertebrates. Immature salmon liver appears to be the richest source of hepatic estrogen receptor so far found for any vitellogenic species.

Animals↗
PubMed1985

Selective inhibitory effects of thyroid hormones on estrogen-induced protein synthesis in chick embryo liver.

We have investigated the effect of thyroid hormones on estrogen-induced responses in embryonic chick liver. Administration of thyroid hormones inhibits estrogen induction of vitellogenin, as well as of apoprotein-II of very low density lipoprotein (VLDL apo-II). A proportionate decrease in the concentration of hepatic salt-soluble nuclear estrogen receptor is also observed. In contrast, estrogen stimulation of apoprotein-B (VLDL apo-B) synthesis is relatively resistant to inhibition. The inhibitory effects of the thyroid hormones could be due to increased metabolism and clearance of estradiol-17 beta in their presence. The relative resistance of estrogen-induced VLDL apo-B synthesis to thyroid hormone inhibition can be explained by its greater sensitivity to low doses of estradiol. In addition, experiments with the antithyroid agent thiourea suggest that, in vivo, estrogen-induced responses could be balanced by the selective inhibitory effects of thyroid hormones.

Animals↗
PubMed1984

Independent developmental programs for two estrogen-regulated genes.

Measurement of hepatic apolipoprotein II and vitellogenin II messenger RNA during chicken embryogenesis showed that these genes acquire estrogen responsiveness at different stages of development. Sensitive solution hybridization assays with excess complementary DNA showed that apolipoprotein II transcripts were induced to 500 molecules per cell at day 9, whereas induction of vitellogenin II messenger RNA was not found until day 11. Thus, two estrogen regulated genes of common function and coordinately regulated in the adult may be on independent developmental programs.

Animals↗
PubMed1984

The effect of molybdate on the intracellular distribution of estrogen receptor in mammary tumors.

The addition of sodium molybdate (20 mM) to human breast tumor homogenates results in an increased yield of estrogen receptor sites in the cytosol, but reduces the number of sites detectable in salt extracts of purified nuclei. Only about 25% of the increase in cytosol receptor can be accounted for by the loss of nuclear sites. Addition of molybdate to isolated nuclear salt extracts has no inhibitory effect on the estrogen receptor assay, but addition of the oxyanion to the cytosol after separation of the initial nuclear pellet still gives a significant increase in estradiol binding capacity. Furthermore, addition of molybdate to the suspension from the first nuclear pellet still results in loss of binding sites in the nuclear salt extract. This rules out the possibility that the negative effect of molybdate on nuclear receptor recovery is by its inhibition of cytosol receptor activation and translocation during tissue preparation. The number of nuclei isolated from estrogen receptor-containing tumors does not differ significantly in the presence or absence of molybdate. Estrogen receptor-negative tumors however yield fewer nuclei, particularly when they are processed in the presence of molybdate. We conclude that when homogenization of tumors is carried out in the presence of molybdate, a significant fraction of estrogen receptor which would normally be present in purified nuclei is lost, perhaps by leakage through the nuclear membrane.

Breast Neoplasms↗
PubMed1984

Hybridization of DNA to RNA in methylmercuric hydroxide agarose gels.

We describe a method for hybridization of cDNA probes to RNA directly in agarose gels which provides a practical alternative to methods involving transfer of the RNA out of the gel. Total cellular RNA is subjected to electrophoresis in agarose gels containing methylmercuric hydroxide as the denaturing agent. After removal of the methylmercuric hydroxide, the gel is dried and 32P-labeled DNA probes are hybridized to the immobilized RNA. This method is more economical in time and expense than methods involving transfer of the RNA out of the gel, while maintaining a level of sensitivity comparable to other procedures.

Animals↗
PubMed1984

Triphenylethylene antiestrogen-binding sites in cockerel liver nuclei: evidence for an endogenous ligand.

Salt extracts of purified nuclei from cockerel liver contain a limited number of sites that bind triphenylethylene nonsteroidal antiestrogens with high affinity and specificity. The assay of the [3H]tamoxifen (3H-labeled 1-[4-(2-dimethylaminoethyoxy)phenyl] 1,2-diphenylbut-1-(Z)ene)-binding sites is optimally achieved by preincubation of the salt extracts with charcoal-dextran suspension; a 4- to 8-fold increase in activity over that obtained with nontreated extracts is found. This suggests that the binding sites are occupied in vivo by an unknown endogenous ligand. The equilibrium dissociation constant for [3H]tamoxifen binding is 4.76 +/- 1.8 nM, and the binding site concentration is 1.7 +/- 0.7 fmol/microgram DNA. The concentration of high affinity estrogen-binding sites in the same extracts is almost 30-fold less (0.06 +/- 0.01 fmol/micrograms DNA). The relative binding affinities of various antiestrogens for the nuclear antiestrogen-binding sites (with tamoxifen arbitrarily set at 100%) are as follows: nafoxidine (1-[2-(p-[3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl]phenoxy)ethyl] pyrrolidine hydrochloride); 126%) greater than tamoxifen (100%) greater than N-des-methyltamoxifen (16%) greater than CI-628 (alpha-[p-[2-(1-pyrrolidine)ethyoxy]phenyl] 4-methoxy-alpha'-nitrostilbene; 14%) greater than 4-hydroxytamoxifen (7%). Estrogens (17 beta-estradiol, estriol, estrone, and diethylstilbestrol) and several other steroids (cholesterol, dihydrotestosterone, pregnenolone, progesterone, and hydrocortisone) show little or no affinity for binding to the nuclear sites (relative binding affinity, less than 0.5%). However, ether extracts of cockerel serum or liver nuclei contain a substance(s) that competitively inhibits [3H]tamoxifen binding to the nuclear antiestrogen-binding sites. The ether-soluble material does not compete for [3H]estradiol binding to the salt-soluble nuclear estrogen receptor. These studies suggest that cockerel serum and liver nuclei contain a natural compound which competes with the triphenylethylenes at the antiestrogen-binding site and may occupy the nuclear binding sites in vivo.

Animals↗
PubMed1982

High affinity binding of anti-oestrogen to the chick liver nuclear oestrogen receptor.

Tamoxifen is a potent inhibitor of specific oestrogen-induced yolk protein synthesis by chicken liver. The oestradiol receptor in salt extracts of liver nuclei from oestrogen-treated chicks has a K(d) for oestradiol of 0.7+/-0.2nm. Tamoxifen and its metabolite, monohydroxytamoxifen, compete for binding to the salt-soluble nuclear receptor with K(i) values of 2.6 and 0.1nm respectively. The anti-oestrogens show much less inhibition of [(3)H]oestradiol binding when assays are carried out using intact nuclei. The competition by unlabelled oestradiol for [(3)H]oestradiol binding to receptor is identical in both salt extracts and intact nuclei. This suggests that intact nuclei contain components which bind anti-oestrogens, but not oestradiol. While tamoxifen and desmethyltamoxifen will readily dissociate from the salt-soluble nuclear oestrogen receptor, monohydroxytamoxifen does not dissociate under the conditions generally used for exchange assays. A modified assay was developed in which 60-70% of monohydroxytamoxifen-bound sites were shown to be exchangeable for [(3)H]oestradiol. Soluble receptor preparations were first incubated in a 1.7% charcoal suspension at 37 degrees C for 15min before assay of specific oestradiol binding. This technique was used in examining the effects of tamoxifen and monohydroxytamoxifen given in vivo on the nuclear oestrogen receptor concentration. Despite their 30-fold difference in binding affinity for the receptor, both anti-oestrogens increase nuclear receptor levels to about the same degree. When given with oestradiol, both compounds have the same apparent partial inhibitory effect on the oestrogen-induced increase in nuclear receptor. These data are consistent with the metabolic hydroxylation of tamoxifen before binding to the hepatic oestrogen receptor.

Animals↗
PubMed1981

Estrogen receptor and the development of estrogenic responses in embryonic chick liver.

We have examined the development of responsiveness to estrogen by embryonic chick liver with a view to determining common and unique factors involved in the establishment of different genomic responses to th e hormone. The major apoproteins of chick VLDL, apo VLDL-B and apo VLDL-II, both appear to be estrogen inducible at an earlier stage of of embryonic development than is vitellogenin. Apo VLDL-B, but not vitellogenin, exhibits a significant level of hepatic synthesis in the absence of estrogen treatment. This basal synthesis in the absence of estrogen treatment. This basal synthesis is tamoxifen-resistant and is detectable at very early stages of hepatic development, well before estrogen responsiveness is seen. Immunological cross-reactivity, electrophoretic behavior and the results of limited proteolysis mapping suggest that the apo VLDL-B synthesized under basal and estrogen-stimulated conditions is the same (or a very similar) protein. Inducibility of the VLDL apoproteins appears to parallel the appearance of the hepatic estrogen receptor system at days 10-12 while vitellogenin induction is delayed by several days. Cytosol receptor concentration undergoes a gradual increase up to the 19th day of development and thereafter declines. The properties of the 19-day receptor are very similar to those of cytosol receptor in hatched chickens, but the fall in concentration does not appear to be proportionately related to inducibility of estrogenic responses, as measured by the relative rates of synthesis in vitro.

Animals↗
PubMed1981

Differential ontogeny of estrogen responsiveness in the chick embryo liver.

The development of estrogen responsiveness in embryonic chicken liver has been studied in terms of specific genomic responses to the hormone. Embryonated eggs were injected with estradiol at various stages of development, and after 48 h the hepatic synthesis of the apoproteins II and B of very low density lipoprotein (VLDL) was determined by incubation of liver slices with [3H]leucine and analysis of the tissue supernatant by specific immunoprecipitation. Significant estrogen induction of synthesis of apoprotein II of VLDL could be seen by the 11th day of embryonic development. The induction of apoprotein B, the other major apolipoprotein of VLDL, paralleled that of apoprotein II. It was reported earlier that the synthesis of vitellogenin did not become estrogen inducible until about the 15th day of embryonic development. The dissociation of the vitellogenin and apoprotein B responses can be confirmed by direct electrophoretic analysis of [3H]serine-labelled liver homogenates. The results are discussed in relation to the ontogeny of the estrogen receptor and the possible involvement of other controlling factors.

Animals↗
PubMed1980

Synthesis of apoprotein B of very low density lipoprotein in embryonic-chick liver. Development of responsiveness of oestrogen.

The influence of exogenous oestrogen on the rate of synthesis of the apoprotein B of very-low-density lipoprotein by embryonic chick liver has been examined at various stages of development. Significant synthesis of apoprotein B was found in the absence of hormone treatment as early as day 6 of egg incubation. This basal level of apoprotein B synthesis varied from 2.5--6% of total protein synthesis. Embryos at day 10 or earlier treated with oestradiol exhibited no increase in the basal level of apoprotein B synthesis. Embryos at day 11 responded to oestradiol slightly and, from days 12 to 20, oestrogen treatment raised the relative rate of apoprotein B synthesis significantly above basal values: the maximal rate was about 16% of total protein synthesis. The anti-oestrogen tamoxifen citrate abolished the hormone-induced increase in apoprotein B synthesis and was not itself oestrogenic. The basal level of apoprotein B production was not sensitive to tamoxifen citrate, either in relatively early or in later stages of development. The basal level of apoprotein B synthesis, therefore, is oestrogen-independent and under developmental control distinct from the hormone-sensitive synthesis. The ontogeny of oestrogen-responsiveness of apoprotein B production appears to parallel the acquisition of the hepatic oestrogen-receptor system [Lazier (1978) Biochem. J. 174, 143--152].

Animals↗