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R L Sutherland

Publications and source records attributed to R L Sutherland.

At least 163 records · Page 9Linked to original sources
PubMed1983

Tamoxifen induces accumulation of MCF 7 human mammary carcinoma cells in the G0/G1 phase of the cell cycle.

When MCF 7 human mammary carcinoma cells in exponential growth phase were treated with tamoxifen a dose-dependent inhibition of cell growth was observed. This inhibition was accompanied by a dose-dependent decrease in the percentage of S phase cells and a concomitant increase in the percentage of cells in the G0/G1 phase of the cell cycle. Simultaneous treatment of cultures with a 10-fold lower concentration of oestradiol completely reversed the growth inhibitory and cell cycle effects of tamoxifen at doses below 5 microM but only partially reversed the effects of higher doses of this drug. It is concluded: (1) that tamoxifen-induced growth inhibition is associated with major changes in the cell cycle kinetic parameters of MCF 7 cells, indicating that this drug is a cell cycle phase-specific growth inhibitory agent and (2) that not all the anti-proliferative and cell cycle effects of tamoxifen in vitro are reversed by simultaneous treatment with oestradiol. This suggests that tamoxifen, in addition to having effects on cell proliferation that are reversed by oestrogens and are likely to be oestrogen receptor-mediated, has antitumor activity in vitro that involves biochemical mechanisms independent of the oestrogen receptor system.

Breast Neoplasms↗
PubMed1983

The interaction of estrogen- and antiestrogen-receptor complexes with polynucleotides.

As the polynucleotide domain of the estrogen receptor (Re) is a possible site for the modulation of Re activity, the interaction of antiestrogen (4-hydroxytamoxifen and tamoxifen)-receptor complexes (4-OH-Tam-Re and Tam-Re) with polynucleotides (oligodeoxynucleotide-cellulose, DNA-cellulose, and polyribonucleotide-agarose) was investigated and compared with that of the 17 beta-estradiol-receptor complex (E2-Re). E2-Re- and anti-Re-E2-complexes were optimally bound to oligo(dT)-cellulose and poly(U)-agarose at 0.15 M KCl and pH 7.6. Temperature activation of the Re was not required for these selective interactions to occur, but they were inhibited by 10 mM sodium molybdate. OH-Tam-Re and E2-Re demonstrated similar selectivity for different deoxynucleotide bases [oligo(dG) greater than oligo(dT) greater than or equal than oligo(dC) greater than oligo(dA) greater than or equal to oligo(dI)] and different ribonucleotide bases [poly(G) = poly(I) greater than poly(U) greater than poly(A) greater than or equal to poly(C)]. Quantitatively, Tam-Re bound significantly less to oligo(dT)-cellulose than did OH-Tam-Re, which did not differ significantly from E2-Re, a result probably related to the dissociation of the lower affinity ligand from the Re/oligo(dT)-cellulose during the assay procedure. Unoccupied Re also bound selectively to these synthetic polynucleotides. It is concluded that: 1) selective binding of Re to synthetic polynucleotides is inhibited by 10 mM sodium molybdate; 2) estradiol is not essential for selective binding to synthetic polynucleotides, since unoccupied Re was only slightly less efficiently bound than E2-Re; and 3) selective binding of Re to synthetic polynucleotides was unaffected by the agonist or antagonist properties of the ligand bound at the steroid-binding site. These data suggest that nonsteroidal antiestrogens are unlikely to exert their antagonist activity by modulating the function of the polynucleotide-binding site of Re.

Animals↗
PubMed1983

Antitumor activity of clomiphene analogs in vitro: relationship to affinity for the estrogen receptor and another high affinity antiestrogen-binding site.

The antitumor activity of three enclomiphene (trans-1- (p-beta-diethylaminoethoxyphenyl)1,2-diphenyl-2-chloroethylene) analogs and the cis isomer zuclomiphene was investigated in MCF 7 human mammary carcinoma cells in culture. Relative antitumor activity was compared with relative binding affinities (RBAs) for the nuclear estrogen receptor (RE; estradiol = 100%) and a microsomal antiestrogen-binding site (AEBS; tamoxifen = 100%) measured in cell-free extracts from MCF 7 cells. Modifications in the structure of the diethylaminoethoxy side chain influenced affinity of both RE and AEBS. Deethylation of the side chain (9599) reduced affinity for both sites by 65-70%, while a diethylaminoproproxy side chain (6866) resulted in a 3-fold increase in affinity for RE (enclomiphene = 2%; 6866 = 6%), but reduced the RBA for AEBS by 66% (enclomiphene = 140%; 6866 = 45%). Conversion of the ether linkage to an amine (10222) increased affinity for RE (10222 = 5%), but markedly reduced affinity for the AEBS (10222 = 15%). All five compounds inhibited the growth of MCF 7 cells in culture, but with markedly different potencies. At low doses (0.25-1.0 microM), where the growth-inhibitory effects were reversed by estradiol (except in the case of zuclomiphene), the relative antitumor activity (6866 greater than 10222 greater than enclomiphene greater than 9599 greater than zuclomiphene) was in the same order as RBA for RE. At higher doses (greater than 2.5 microM), where the growth-inhibitory effects were unaffected or partially reversed by estradiol, the relative potencies of these compounds as antitumor agents changed. Zuclomiphene became the most active, while the potency of enclomiphene increased relative to 6866 and 10222 (zuclomiphene greater than enclomiphene greater than 6866 greater than 10222 greater than 9599). At these doses, estrogen-irreversible antitumor activity was unrelated to affinity for RE, but showed some correlation with affinity for AEBS. It is concluded that the major effect of these compounds on mammary carcinoma growth in vitro is an estrogen-reversible growth-inhibitory effect, the magnitude of which is correlated with affinity for RE. However, studies with zuclomiphene and estrogen-irreversible doses of enclomiphene and 6866 indicate that these antiestrogens also influence cell proliferation in vitro by mechanisms that probably do not involve RE.

Antineoplastic Agents↗
PubMed1983

Cell proliferation kinetics of MCF-7 human mammary carcinoma cells in culture and effects of tamoxifen on exponentially growing and plateau-phase cells.

MCF-7 human mammary carcinoma cells were inoculated into 150-sq cm flasks at 3 X 10(5) cells/flask, and after a lag period of about 48 hr, these cells grew exponentially for 5 days with a mean population doubling time of about 24 hr. During exponential growth, 80 to 90% of cells were in the "rapidly cycling" pool, the clonogenic fraction was 50 to 60%, and the mean percentage of cells in the G0-G1, S, and G2 + M phases of the cell cycle was 48.9 +/- 0.6% (S.E.), 39.4 +/- 0.6%, and 11.6 +/- 0.3%, respectively. These parameters changed rapidly between Days 7 and 13 when plateau phase was reached. Between Days 13 and 18, 74.8 +/- 0.7% of cells were in G0-G1, 15.3 +/- 0.4% were in S, and 9.8 +/- 0.6% were in G2 + M phase. Only about 30% of these cells were cycling rapidly, and the clonogenic fraction had fallen to less than 10%. Tamoxifen induced a dose-dependent decrease in the growth rate of exponentially growing cells, which was accompanied by a dose-dependent increase in percentage of G0-G1-phase cells, and a decline in percentage of S-phase cells. At doses greater than or equal to 10 microM, a 24-hr pulse of tamoxifen was cytotoxic to exponentially growing cells. Plateau-phase cells were less sensitive to these effects of tamoxifen. In an attempt to define the kinetic basis of the G0-G1 accumulation induced by tamoxifen, asynchronous MCF-7 cells were pretreated for 42 hr with various doses of tamoxifen, and the rate of efflux of cells from the G0-G1 phase of the cell cycle was assessed by blocking their reentry into G1 with ICRF 159. Following treatment of control cultures with ICRF 159, two populations of cells were distinguished by their rates of efflux from G0-G1 phase. The majority of cells left G0-G1 rapidly with a mean t1/2 of 2.3 hr ("rapidly cycling" cells). However, about 18% of cells had a much slower rate of exit with a mean t1/2 of about 30 hr ("slowly cycling" cells). Pretreatment with tamoxifen resulted in a dose-dependent decrease in the proportion of rapidly cycling cells and an increase in the proportion of cells with slow G1 transit times. Although this appeared to be the predominant effect, tamoxifen also decreased the rate at which the slowly cycling cells traversed G1. Simultaneous treatment with estradiol returned these parameters to control values at doses of tamoxifen less than or equal to 5 microM, partially reversed the effect of 7.5 microM tamoxifen, but was without effect on the arrest of cell cycle progression induced by 10 microM tamoxifen. It is concluded that cells accumulate in G0-G1 following tamoxifen treatment due to an increase in the proportion of slowly cycling cells at the expense of a population of rapidly cycling cells, which appear to be relatively uninfluenced by the drug.

Breast Neoplasms↗
PubMed1983

Effects of tamoxifen on cell cycle progression of synchronous MCF-7 human mammary carcinoma cells.

The effects of tamoxifen on cell cycle progression and clonogenic survival have been examined using synchronized cultures of MCF-7 human mammary carcinoma cells. Cell synchrony was induced by mitotic selection. Subsequent cell cycle analyses, using DNA flow cytometry, showed that 85% of synchronized cells had a mean cell cycle time of 21.3 hr with mean phase durations of 9 hr for G0-G1, 9.3 hr for S, and 3 hr for G2 + M. A slowly cycling or noncycling subpopulation comprising 15% of the total population was also observed. Exposure to tamoxifen (5 to 12.5 microM) resulted in a dose-dependent reduction in the number of cells progressing through G0-G1 and entering S phase. Those cells which were not retained in G0-G1, however, appeared to traverse G0-G1 and the remainder of the cell cycle at a rate only slightly less than that of untreated controls. Further experiments demonstrated that the major sensitivity to tamoxifen in terms of both inhibition of cell cycle progression and drug cytotoxicity was restricted to a short interval in the middle of G0-G1. This 2- to 4-hr period of maximum drug sensitivity began approximately 4 hr after mitotic selection, with drug exposures outside this time frame having markedly fewer effects. The significance of these observations in the light of previous studies with asynchronous populations of MCF-7 cells is discussed.

Breast Neoplasms↗
PubMed1983

Effects of biologically active metabolites of tamoxifen on the proliferation kinetics of MCF-7 human breast cancer cells in vitro.

The effects of two major metabolites of tamoxifen, N-demethyltamoxifen (DMT) and 4-hydroxytamoxifen (4OHT), on MCF-7 cell proliferation and cell cycle kinetic parameters were compared with those of the parent compound. All three compounds produced dose-dependent decreases in the rate of cell proliferation which were accompanied by decreases in the percentage of S- and G2-M-phase cells. 4OHT was 100- to 167-fold more potent than both tamoxifen and DMT in producing these effects, and this was correlated with their relative binding affinities (RBAs) for the cytoplasmic estrogen receptor (ER) (17 beta-estradiol = 100, 4OHT = 41, tamoxifen = DMT = 2). At doses less than or equal to 2.5 microM, these effects were completely reversed by 17 beta-estradiol, but the required 17 beta-estradiol:antiestrogen concentration ratios differed, i.e., 1:10 to 1:1 for 4OHT compared with 1:1000 to 1:100 for tamoxifen and DMT. Although the concentrations of 17 beta-estradiol required for reversal were related to affinity of the metabolite for ER, they were 5- to 20-fold lower than predicted from the measured RBAs. When the rate of cell proliferation was measured over a range of concentrations of antiestrogen, in the presence or absence of 17 beta-estradiol, it was highly correlated (r2 = 0.96) with the percentage of S-phase cells. In addition to these 17 beta-estradiol-reversible events, all three compounds caused 17 beta-estradiol-irreversible cytotoxicity at higher concentrations (greater than or equal to 7.5 microM DMT and 4OHT, 10 microM tamoxifen). The order of potency in producing this effect was DMT greater than 4OHT greater than tamoxifen, which correlated with neither the RBAs for ER nor the RBAs for the high-affinity microsomal antiestrogen binding site. These data support the concept that estrogens and antiestrogens compete for a common event which regulates the rate of cell proliferation probably by controlling the proportion of cells entering S phase. Although it appears likely that ER is intimately involved in this regulatory process, 17 beta-estradiol-irreversible mechanisms are also involved in antiestrogen action in vitro.

Biotransformation↗
PubMed1981

Macropodid marsupial testicular gonadotrophin receptors and their use in assays for marsupial gonadotrophins.

The interactions of several mammalian follicle-stimulating hormones and luteinizing hormones with specific gonadotrophin receptors in macropodid marsupial testicular homogenates were investigated with a view to developing radioreceptor assays for marsupial FSH and LH. Testes from Eastern grey kangaroos and tammar wallabies possessed high affinity (dissociation constant congruent to 10(-10) mol/l) saturable receptor sites which were highly specific for LH or FSH. Luteinizing hormone receptor sites bound only highly purified LH preparations (human, ovine and rat) but did not bind highly purified FSH, TSH or prolactin while FSH receptor sites were equally specific for highly purified FSH preparations. These sites demonstrated a degree of species specificity in that marsupial pituitary extracts were relatively more potent in these assays than in assays using gonadotrophin receptors from rat testes. Serum from hypophysectomized female tammar wallabies had little effect on the slope and position of the LH standard curve but significantly depressed the dose-response curve for FSH. For this reason it was not possible to develop a radioreceptor assay for serum FSH using marsupial testicular FSH receptors. However, gonadotrophin receptors from both rat and marsupial testes have been employed in the successful development of radioreceptor assays for marsupial pituitary LH and FSH and marsupial serum LH.

Animals↗
PubMed1981

A high-affinity binding site for the antioestrogens, tamoxifen and CI 628, in immature rat uterine cytosol which is distinct from the oestrogen receptor.

A high-affinity, saturable antioestrogen binding site, which does not bind oestradiol, has been reported to exist in a number of oestrogen target tissues but not in the immature rat uterus. This study reports the results of a more thorough search for this site in immature rat uterine cytosol. When concentrations of uterine cytoplasmic receptor were selectively depleted by translocation of 90-95% of the cytoplasmic oestrogen receptor to the nucleus, saturation analysis studies revealed that the antioestrogens, tamoxifen and CI 628, were bound to high-affinity, saturable binding sites which were present at about 2.5 times the concentration of the residual oestrogen receptor sites. Oestradiol could only partially inhibit the binding of tritiated antioestrogens to their saturable binding sites in this material indicating that a significant proportion of these sites were distinct from the oestrogen receptor sites. This was confirmed in experiments where oestrogen receptor sites were saturated in vitro with oestradiol and high-affinity, saturable sites for CI 628 and tamoxifen were still present. The CI 628 and tamoxifen had high affinity for these sites with dissociation constants of 1.0-1.6 nmol/l. These specific antioestrogen binding sites were present at about 5% of the concentration of oestrogen receptors in normal immature rat uterine cytosol which probably explains their previous lack of detections in this material.

Animals↗
PubMed1980

High-affinity anti-oestrogen binding site distinct from the oestrogen receptor.

Non-steroidal anti-oestrogens such as tamoxifen, CI 628, nafoxidine and clomiphene, are structurally related synthetic compounds that antagonize the effects of oestrogen on its target tissues, and this activity has led to the use of tamoxifen to treat advanced breast cancer. All these compounds inhibit the binding of tritiated oestradiol to cytosol from oestrogen target tissues, suggesting that anti-oestrogens bind to the oestrogen receptor. This is supported by reports that in the rat uterus and dimethyl-benz (alpha)-anthracene (DMBA)-induced rat mammary carcinoma, oestradiol and anti-oestrogens bind directly to the same number of saturable binding sites. Furthermore, oestrogens and anti-oestrogens are mutually competitive for binding to these sites. It has thus been generally accepted that the anti-oestrogens exert most of their effects through the specific oestrogen receptor. We now report a further high-affinity, anti-oestrogen binding site which may have a role in regulating the effects of non-steroidal anti-oestrogens.

Animals↗
PubMed1980

Steroid receptors and effects of oestradiol and progesterone on chick oviduct proteins.

After a single injection of oestradiol benzoate (1.5 mg/kg) to oestrogen-withdrawn chickens, there was an increase in magnum wet weight, DNA polymerase alpha activity, adenosine-3',5'-monophosphate-dependent protein-kinase activity and estrogen-receptor concentration, as measured over 36 h. Besides these intracellular proteins, the secretory proteins ovalbumin and conalbumin were also augmented, and detailed time-course studies were performed. Early induction kinetics for ovalbumin and conalbumin synthesis, which differed for each protein, were independent of the dose of oestradiol benzoate injected if it exceeded 0.1 mg/kg. After 6 h for ovalbumin and 2 h for conalbumin, the induction curves diverged according to the dose of hormone administered and in correlation with the persistence of elevated nuclear oestrogen-receptor concentrations, a result confirmed with 11 beta-methoxy-17 alpha-ethynyloestradiol (R 2858), a powerful synthetic oestrogen. When oestradiol benzoate (1 mg/kg) and progesterone (3 mg/kg) were injected simultaneously, the rate of conalbumin sythesis, during the first 6-8 h, was lower than that observed in animals injected with oestradiol benzoate alone. However at later times conalbumin synthesis was greater in animals receiving both hormones than with oestradiol alone. In contrast, the rate of ovalbumin synthesis after the combined injection was higher than that induced by either hormone alone throughout the entire experimental period. In order to study further the synergistic and antagonistic activities of these two hormones, a single injection of progesterone (3 mg/kg) was administered 6, 12 or 18 h after 1.5 mg/kg oestradiol benzoate. Progesterone administration resulted in a reduction in cytoplasmic, nuclear and total oestrogen receptor concentration for at least 6 h when compared with the values in birds treated with oestrogen alone. DNA polymerase and protein kinase activities were also reduced during this period. Subsequently, all parameters increased, and by 18-24 h after progesterone treatment, reached values higher than those observed in animals receiving oestrogen alone.

Animals↗
PubMed1980

Macropodid marsupial luteinizing hormone: validation of assay procedures and changes in concentrations in plasma during the oestrous cycle in the female tammar wallaby (Macropus eugenii).

A heterologous double antibody radioimmunoassay employing a rabbit anti-ovine LH antiserum (GDN no. 15) has been developed for the assessment of concentrations of LH in macropodid marsupial pituitary extracts and plasma. In this radioimmunoassay system highly purified ovine, rat human and kangaroo LH preparations demonstrated apparently parallel dose-response curves, as did serial dilutions of crude pituitary extracts from a wide range of Austrlian marsupial species and serial dilutions of plasma from ovariectomized, oestrous and LH releasing hormone (LH-RH)-treated marsupials. The assay has been used to monitor changes in immunoreactive LH in the plasma of the female tammar wallaby. Basal concentrations of LH in non-oestrous female wallabies were in the range < 0.20--1.90 ng/ml with many animals having values at or near the limit of detection of the assay. Concentrations of LH were markedly increased following ovariectomy (1.7--7.0 ng/ml), on the day of oestrus (10.0-- > 50 ng/ml) and following administration of LH-RH (9.5-- > 25.0 ng/ml). Plasma from hypophysectomized animals had no detectable LH immunoactivity, A well-defined LH surge, lasting approximately 12 h, was associated with oestrus. Mating occurred approximately 8 h before, and ovulation approximately 24 h after, the maximal concentrations of LH in plasma.

Animals↗
PubMed1980

Control of gonadotrophin secretion in the female tammar wallaby (macropus eugenii).

A heterologous radioimmunoassay for tammar wallaby FSH, using an ovine FSH antiserum and a human FSH tracer, is described. With this assay concentrations of FSH in plasma of intact female tammars are not detectable except rarely at the time of oestrus. However the assay has proved useful in studies of the control of gonadotrophin secretion in intact male and in ovariectomized tammars. In the female tammar, concentrations of LH and FSH in plasma rose within a few days of bilateral ovariectomy. Ovariectomized tammars respond to a luteinizing hormone releasing hormone stimulus (10 microgram, i.v.) with a prompt release of LH, peak levels of 16.9 +/- 1.4 ng NIH-LH-S19/ml plasma (n = 12) being reached within 25 min of injection. Concentrations of LH and FSH in plasma were reduced to preoperative values in ovariectomized tammars when lutein tissue developed in ovarian cortex grafts autotransplanted under the pouch skin. Ovarian interstitial tissue was not necessary for this effect. After lutectomy during quiescence, the female tammar ovulates again in about 14 days. Injections of progesterone (700 microgram/kg per day, i.m.) for 7 days after the operation did not delay this response, but follicular development and ovulation appeared to be retarded in animals given oestradiol-17 beta (5 microgram/kg per day, i.m.) with or without progesterone.

Animals↗
PubMed1980

Effect of gonadectomy, season and the presence of female tammar wallabies (Macropus eugenii) on concentrations of testosterone, luteinizing hormone and follicle-stimulating hormone in the plasma of male tammar wallabies.

Concentrations of FSH, LH and testosterone in plasma were measured in groups of adult male tammar wallabies before and after gonadectomy, and during the breeding and non-breeding seasons. Gonadectomy resulted in a rapid fall in plasma testosterone to undetectable levels by day 2, and significant increases in plasma LH and FSH levels. The concentrations of FSH, LH and testosterone did not change significantly between the non-breeding and breeding seasons in groups of male wallabies maintained in the absence of females. However, when male wallabies were associated with sexually mature females there were significant three- to fourfold increases in concentrations of LH and testosterone in plasma at the commencement of the breeding season. The observed increases in LH and testosterone were highly synchronized in the eight animals studied and occurred approximately 2 weeks before the synchronous onset of mating. Concentrations of FSH did not change significantly at this time.

Animals↗