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A E Wakeling

Publications and source records attributed to A E Wakeling.

At least 37 records · Page 2Linked to original sources
PubMed1995

MCF-7 human mammary adenocarcinoma cell death in vitro in response to hormone-withdrawal and DNA damage.

Anti-oestrogens exert a tumoristatic effect on estrogen-receptor-positive breast carcinomas in vivo. At a cellular level this may reflect inhibition of cell proliferation and/or cell death counterbalanced by continued proliferation of a cell subpopulation. We evaluated the MCF-7 human mammary adenocarcinoma cell line as an in vitro model to study the effects of the novel oestrogen antagonist ICI 182,720 on cell population dynamics (cell gain vs. cell loss). After oestrogen-withdrawal monolayer cell number declined over 10 days, accompanied by cell detachment. This decrease in viable cell number was elevated 2-fold by ICI 182,780. Detached cells exhibited DNA fragments of 50 and 300 kbp, typical of apoptotic cells. However, internucleosomal cleavage to 180 bp integer fragments was not seen, and these detached cells exhibited a morphology which was not consistent with apoptosis. The remaining attached monolayer cells were morphologically viable (> 99%) with regard to both nuclear morphology and plasma membrane integrity. There was no difference in cell cycle phase distribution between oestrogen-withdrawn and ICI 182,780-treated cells; both induced accumulation in G1 phase. MCF-7 cells were also exposed to a variety of DNA damaging agents known to induce apoptosis in other cell types. We could demonstrate only limited induction of morphologically recognisable apoptosis in MCF-7 cells treated with methyl methanesulphonate. Our results add to the controversy surrounding the ability of the MCF-7 cell line to undergo apoptosis in vitro in response to anti-oestrogen therapies.

Adenocarcinoma↗
PubMed1994

The importance of tamoxifen metabolism in tamoxifen-stimulated breast tumor growth.

The acquired ability of tamoxifen to stimulate tumor growth has been suggested as one mechanism for the development of treatment failure in breast cancer. We have reported that tamoxifen-stimulated MCF-7 breast tumors in nude mice display reduced tamoxifen levels as compared with tamoxifen-inhibited tumors and an altered metabolite profile with isomerization of trans-4-hydroxytamoxifen to a weak antiestrogen and the production of metabolite E, an estrogenic metabolite. To investigate further the importance of tamoxifen metabolism in this model, we quantified levels of tamoxifen and major metabolites in tamoxifen-stimulated as compared with tamoxifen-inhibited MCF-7 tumors growing in nude mice and employed tamoxifen analogs resistant to metabolism. Tamoxifen-stimulated tumors have a relative abundance of cis-4-hydroxytamoxifen and metabolite E. However, in vivo treatment of mice carrying tamoxifen-stimulated tumors with fixed-ring nonisomerizable tamoxifen analogs or with nafoxidine, a nonsteroidal antiestrogen with a different structure, nonetheless resulted in tumor growth stimulation. Tumors were also stimulated by a deoxytamoxifen analog resistant to conversion to metabolite E. Growth of tamoxifen-stimulated tumors was inhibited by a pure steroidal antiestrogen, ICI 182,780, suggesting the need for clinical trials of this drug in patients with tamoxifen resistance. Growth of tamoxifen-stimulated tumors was further stimulated by estrogen replenishment, and this estrogen stimulation could be blocked by tamoxifen indicating that tamoxifen has both agonist and antagonist properties in these tumors. This study suggests that tamoxifen-stimulated tumor growth in this model is not due to isomerization or metabolism of tamoxifen to less antiestrogenic or more estrogenic metabolites. The mechanisms by which tamoxifen acquires more potent in vivo agonist properties, resulting in tumor growth stimulation over time, remain to be defined.

Animals↗
PubMed1994

Effects of a non-steroidal pure antioestrogen, ZM 189,154, on oestrogen target organs of the rat including bones.

ZM 189,154 ([1RS,2RS]-2-(4-hydroxyphenyl)-2-methyl- 1-[9-(4,4,5,5,5-penta-fluoropentyl)sulphinylnonyl]-1,2,3,4- tetrahydronaphth-6-ol) is a non-steroidal pure antioestrogen. It has a high relative affinity for the oestrogen receptor, completely blocks the trophic action of oestradiol (OE2) on the uterus in immature and ovariectomized (OVX) adult rats and, in the latter, also completely blocks the trophic action of OE2 on vagina, bone and growth rate. ZM 189,154 displays no intrinsic oestrogen-agonist activity on uterus, vagina, bone, LH secretion or growth rate in OVX rats. Differential sensitivity of OE2-regulated processes was more apparent in intact rats. Daily doses of 0.6 mg/kg per day of ZM 189,154 blocked ovulation; 2 mg/kg per day achieved maximal uterine atrophy but did not affect bone density or growth rate; 10 mg/kg per day produced a broader spectrum of effects (reduced bone density, increased basal LH, slightly increased growth rate), but the magnitude of these was smaller than after ovariectomy; the 10 mg/kg dose also produced multiple ovarian follicular cysts. The failure of ZM 189,154 to achieve complete ovariectomy-like effects in intact rats may be due to the action of ovarian factors other than OE2, or to the circulating OE2 levels resulting from the disturbance to ovarian function posing too strong a challenge to the antagonist.

Animals↗
PubMed1993

MCF7/LCC2: a 4-hydroxytamoxifen resistant human breast cancer variant that retains sensitivity to the steroidal antiestrogen ICI 182,780.

The development of resistance to the antiestrogen tamoxifen occurs in a high percentage of initially responsive patients. We have developed a new model in which to investigate acquired resistance to triphenylethylenes. A stepwise in vitro selection of the hormone-independent human breast cancer variant MCF-7/LCC1 against 4-hydroxytamoxifen produced a stable resistant population designated MCF7/LCC2. MCF7/LCC2 cells retain levels of estrogen receptor expression comparable to the parental MCF7/LCC1 and MCF-7 cells. Progesterone receptor expression remains estrogen inducible in MCF7/LCC2 cells, although to levels significantly lower than observed in MCF-7 and MCF7/LCC1 cells. MCF7/LCC2 cells form tumors in ovariectomized nude mice without estrogen supplementation, and these tumors are tamoxifen resistant but can be estrogen stimulated. Significantly, MCF7/LCC2 cells have retained sensitivity to the steroidal antiestrogen ICI 182,780. These data suggest that some breast cancer patients who acquire resistance to tamoxifen may not develop cross-resistance to treatment with steroidal antiestrogens.

Animals↗
PubMed1993

Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation.

To date, apoptosis has been characterized biochemically by the production of 180-200 bp internucleosomal DNA fragments resulting from the activation of an endonuclease(s). The principal morphological feature of apoptosis is the condensation of chromatin and it has been assumed that this may reflect the oligonucleosomal fragmentation pattern. We have re-examined this dogma by comparing the biochemical and morphological features of cell death in several epithelial cell types (HT-29-I1 colon adenocarcinoma, CC164 mink lung, DU-145 human prostatic carcinoma and MCF-7 human breast adenocarcinoma) and one mesenchymal cell line (H11ras-R3 ras-transformed rat fibroblasts). Cell death was induced either by serum deprivation, TGF-beta 1 or etoposide, or by leaving cells to reach confluence. Cell death was assessed with respect to detachment from monolayers, morphological changes and DNA integrity. The DNA-binding fluorophore Hoechst 33258 revealed chromatin condensation patterns consistent with apoptotic cell death in all cell types except MCF-7 cells. Using field inversion gel electrophoresis in conjunction with conventional 2% agarose gel electrophoresis, cleavage of DNA to 50 kbp fragments was observed in all cases except MCF-7 cells. This preceded the appearance of oligonucleosomal fragments in HT-29-I1, CC164 and H11ras-R3 cells. Although the DNA of DU-145 cells fragmented into 50 kbp units, and although the cells exhibited classical apoptotic morphology, no subsequent internucleosomal cleavage was observed. These results suggest that changes in the integrity of DNA indicative of the release of chromatin loop domains occur before cleavage at internucleosomal sites is initiated and that the latter is not an essential step in the apoptotic process.

Animals↗
PubMed1993

The future of new pure antiestrogens in clinical breast cancer.

The rationale for seeking to identify new pure antiestrogens was based on the recognition that existing antiestrogens, exemplified by tamoxifen, all possess partial agonist (estrogenic) activity. Conceptually, pure antiestrogens should be more effective than tamoxifen in ablating the mitogenic action of estrogens on breast tumor growth. The discovery and properties of the pure antiestrogens ICI 164,384 and ICI 182,780 are described and contrasted with those of tamoxifen. Key characteristics of these compounds which may be of particular relevance to their therapeutic application in the treatment of breast cancer are described. These include experimental data which predict efficacy in patients whose disease recurs during tamoxifen treatment, and the potential for pure antiestrogens to demonstrate greater efficacy than tamoxifen in first-line treatment of advanced breast cancer. The data imply that gains in efficacy could emerge as more rapid, more complete, or longer-lasting tumor remissions. Clinical trials with ICI 182,780 will reveal whether one or more of these predictions is correct.

Breast Neoplasms↗
PubMed1993

Type I IGF receptor and acquired tamoxifen resistance in oestrogen-responsive human breast cancer cells.

Tamoxifen inhibited the oestrogen-stimulated proliferation of MCF-7 cells but had little effect on the oestrogen-stimulated proliferation of two tamoxifen-resistant variants (RL-3 and AL-1). The lack of oestrogen antagonist activity in the resistant cells was largely a result of an increased oestrogen agonist activity of tamoxifen on cell proliferation. Proliferation of the tamoxifen-resistant cells was also stimulated by 4-hydroxytamoxifen but not by ICI 164,384, a structurally distinct pure anti-oestrogen. Tamoxifen does not have increased oestrogen agonist activity for the induction of a series of oestrogen-regulated RNAs, and this suggests that the increased agonist activity may be restricted to key components involved in the proliferative response. Tamoxifen-stimulated cell proliferation was dependent on insulin-like growth factor I (IGF-1) in the resistant cells, suggesting that tamoxifen stimulates cell proliferation by sensitising cells to the proliferative effects of IGF-1. This may involve induction of the type-I IGF receptor.

Breast Neoplasms↗
PubMed1993

Are breast tumours resistant to tamoxifen also resistant to pure antioestrogens?

A substantial proportion of patients with breast cancer are treated with the antioestrogen tamoxifen. As with other endocrine therapies, clinical experience has shown that some tumours in which growth is initially attenuated by tamoxifen treatment become resistant to continued drug treatment and resume growth. The mechanisms underlying the development of tamoxifen resistance have yet to be described but represent an important focus of research with the aim of defining what other therapies might be effective following tamoxifen treatment. Secondly, an understanding of tamoxifen resistance might suggest means to develop more effective agents for primary treatment of the disease. The development of pure antioestrogens, for example ICI 164,384 and ICI 182,780, which differ pharmacologically from tamoxifen in being entirely free of oestrogen partial-agonist activity, together with cell and animal models of tamoxifen resistant human breast cancer, has revealed one mechanism which might be of considerable clinical significance. Pure antioestrogens were shown to inhibit the proliferation of a greater proportion of tumor cells than tamoxifen in vitro, a differential effect that was attributed to the oestrogenic activity of tamoxifen. Subsequently, cell culture studies have shown that breast cancer cell lines selected for resistance to tamoxifen can still remain sensitive to the growth inhibitory action of pure antioestrogens. Similarly, the growth of human breast tumours in nude mice, which is initially attenuated by tamoxifen but then resumes, can be inhibited by pure antioestrogens. Both types of experiment are consistent with the view that tamoxifen resistance in these model systems is due to the oestrogenic action of tamoxifen. Thus, it can be predicted that in some patients whose tumours recur during tamoxifen therapy, a further response to pure antioestrogen treatment might occur. Studies to examine this hypothesis are currently being undertaken with ICI 182,780. One mechanism which might account for the experimental observations is an intrinsic heterogeneity amongst breast tumour cells in their response to tamoxifen, i.e. that there are at least two different populations of cells; one population which responds to tamoxifen as an antioestrogen and one which "reads" tamoxifen as an oestrogen. The growth advantage thus conferred on the latter population would lead to its predominance. If this is what actually happens in a proportion of human tumours, it can be argued that primary treatment of the tumour with a pure antioestrogen, rather than tamoxifen, would be preferred since a more complete and longer-lasting response would be predicted. Recent comparative studies with human breast tumours grown in nude mice support these predictions.

Breast Neoplasms↗
PubMed1993

Antiuterotrophic effects of the pure antioestrogen ICI 182,780 in adult female monkeys (Macaca nemestrina): quantitative magnetic resonance imaging.

The antiuterotrophic efficacy of the pure antioestrogen ICI 182,780 has been demonstrated previously by magnetic resonance imaging (MRI) in ovariectomized oestrogen-treated monkeys (Macaca nemestrina). Further characterization of the effects of ICI 182,780 in intact adult female monkeys with normal menstrual cycles was undertaken to provide an indication of its potential actions in premenopausal women. Changes in the volume of uterine tissues were measured by MRI in early, mid and late cycle. The volume of the uterus varied up to fivefold between individual monkeys but serial observations in individuals provided sufficient precision to allow accurate assessments to be made of changes in the endometrium and myometrium during the course of the menstrual cycle and following ICI 182,780 administration. In comparison with its initial size in untreated monkeys, the endometrium increased in volume by 60% and 125% in the mid and late cycle respectively. In contrast, the size of the myometrium decreased significantly, by 16% from early to mid cycle and then recovered to near its initial volume in the late cycle. Treatment with ICI 182,780 beginning in the early part of the menstrual cycle prevented the growth of the uterus. The magnitude and duration of the response was dependent on whether or not ovulation occurred during treatment with ICI 182,780. In animals rendered anovulatory, growth of the endometrium was blocked completely by ICI 182,780 and the volume of the tissue declined below that present at the start of the menstrual cycle. Antiuterotrophic efficacy was significantly less in monkeys which ovulated during treatment with ICI 182,780.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

ICI 182,780, a new antioestrogen with clinical potential.

Previous studies in this laboratory identified a series of 7 alpha-alkylamide analogues of 17 beta-oestradiol which are pure antioestrogens. Among this initial lead series of compounds, exemplified by ICI 164,384, none was of sufficient in vivo potency to merit serious consideration as a candidate for clinical evaluation. Further structure-activity studies identified a new compound, ICI 182,780, 7 alpha-[9-(4,4,5,5,5-pentafluoro-pentylsulphinyl)nonyl]oestra-1,3,5(10)- triene-3,17 beta-diol, with significantly increased antioestrogenic potency. The antiuterotrophic potency of ICI 182,780 is more than 10-fold greater than that of ICI 164,384. ICI 182,780 has no oestrogen-like trophic activity and, like ICI 164,384 is peripherally selective in its antioestrogenic effects. The increased in vivo potency of ICI 182,780 was also reflected, in part, by intrinsic activity at the oestrogen receptor and in the growth inhibitory potency of ICI 182,780 in MCF-7 human breast cancer cells. ICI 182,780 was a more effective inhibitor of MCF-7 growth than 4'-hydroxytamoxifen, producing an 80% reduction of cell number under conditions where 4'-hydroxytamoxifen achieved a maximum of 50% inhibition. Sustained antioestrogenic effects of ICI 182,780, following a single parenteral dose of ICI 182,780 in oil suspension, were apparent in both rats and pigtail monkeys. In vivo, the antitumour activity of ICI 182,780 was demonstrated with xenografts of MCF-7 and Br10 human breast cancers in athymic mice where, over a 1 month period, a single injection of ICI 182,780 in oil suspension achieved effects comparable with those of daily tamoxifen treatment. Thus, ICI 182,780 provides the opportunity to evaluate clinically the potential therapeutic benefits of complete blockade of oestrogen effects in endocrine-responsive human breast cancer.

Animals↗
PubMed1992

Antiuterotrophic effects of a pure antioestrogen, ICI 182,780: magnetic resonance imaging of the uterus in ovariectomized monkeys.

ICI 182,780 is a potent specific pure antioestrogen which may offer advantages in breast cancer treatment compared with partial agonists like tamoxifen. To characterize further the potency and efficacy of ICI 182,780, its effects on the uterus of ovariectomized, oestrogen-treated monkeys (Macaca nemestrina) have been measured using magnetic resonance imaging (MRI). Quantitative MRI allows accurate non-invasive repetitive measurements of endometrial and myometrial volume following hormonal treatments, using each animal as its own control. Single i.m. injections of a long-acting oil-based formulation of ICI 182,780 sustained blockade of oestradiol action on the monkey uterus in a dose-dependent manner for 3-6 weeks. Repeated injections of 4 mg ICI 182,780/kg at 4-weekly intervals provided increasingly effective blockade of uterine proliferation. In a short-acting formulation, ICI 182,780 also completely blocked the trophic action of oestradiol, administered concurrently, in ovariectomized monkeys. Similarly, ICI 182,780 caused involution of the uterus stimulated by prior treatment with oestradiol. The rate and extent of uterine involution in monkeys treated with ICI 182,780 was similar to that seen following oestrogen withdrawal. These studies demonstrate that ICI 182,780 is a fully effective pure antioestrogen in a primate.

Animals↗
PubMed1992

Steroid antagonists as nuclear receptor blockers.

Molecular analysis of sex steroid receptors, together with the availability of a range of ligands for these receptors that affect differentially the physiological actions of the ligand-receptor complex in the cell nucleus, is providing new insights into the processes that control transcriptional activation leading to cell growth. These studies serve to focus attention on the importance of other cell specific factors that are important in determining the transcriptional efficiency of the hormone-receptor complex. Elucidation of the nature and role of such factors may lead ultimately to hormonal or anti-hormonal agents targeted to modulate selected hormone mediated events in specific cells or organs. The sex steroid receptors are prototypes of a much larger family of proteins that control transcription, including proteins that bind to ligands known to be important physiologically, such as thyroid hormones and retinoids, as well as "orphan" receptors for which no ligands have been described. The established value of steroid antagonists in cancer therapy suggests that further developments in the understanding of new members of the steroid receptor superfamily may lead to the development of novel anti-cancer agents. The developing understanding of "cross talk" between steroid-receptor and growth factor-receptor mediated tumour growth stimulatory pathways may also provide previously unrecognized opportunities for tumour therapy.

Androgen Antagonists↗
PubMed1991

A potent specific pure antiestrogen with clinical potential.

Previous studies from this laboratory have described a series of 7 alpha-alkylamide analogues of estradiol with pure antiestrogenic activity, exemplified by ICI 164,384. A new compound, 7 alpha-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)nonyl]estra-1,3,5(10 )- triene-3,17 beta-diol (ICI 182,780) has now been identified which has significantly increased antiestrogenic potency and retains pure estrogen antagonist activity. The antiuterotrophic potency of ICI 182,780 in the immature rat was more than 10-fold greater than that of ICI 164,384 (50% effective doses of 0.06 and 0.9 mg/kg, respectively). This order of magnitude increase of in vivo potency was also reflected, in part, by intrinsic activity at the estrogen receptor. The relative binding affinities of ICI 182,780 and ICI 164,384 were 0.89 and 0.19, respectively, compared with that of estradiol (1.0). Similarly, the in vitro growth-inhibitory potency of ICI 182,780 exceeded that of ICI 164,384 in MCF-7 human breast cancer cells, where 50% inhibitory concentrations of 0.29 and 1.3 nM, respectively, were recorded. ICI 182,780 was a more effective inhibitor of MCF-7 growth than 4'-hydroxytamoxifen, producing an 80% reduction of cell number under conditions where 4'-hydroxytamoxifen achieved a maximum of 50% inhibition. This increased efficacy was reflected by a greater reduction of the proportion of cells engaged in DNA synthesis in ICI 182,780-treated cell cultures compared with tamoxifen-treated cells. Sustained antiestrogenic effects, following a single parenteral dose of ICI 182,780 in oil suspension, were apparent in both rats and pigtail monkeys. In vivo, antitumor activity of ICI 182,780 was demonstrated with xenografts of MCF-7 and Br10 human breast cancers in nude mice. A single injection of ICI 182,780 provided antitumor efficacy equivalent to that of daily tamoxifen treatment for at least 4 weeks. The properties of ICI 182,780 identify this pure antiestrogen as a prime candidate with which to evaluate the potential therapeutic benefits of complete estrogen withdrawal in endocrine-responsive human breast cancer.

Animals↗
PubMed1990

Therapeutic potential of pure antioestrogens in the treatment of breast cancer.

Novel 7 alpha-analogues of 17 beta-oestradiol like ICI 164,384, differ from all antioestrogens described previously in being entirely free of partial agonist activity. In adult rats, ICI 164,384 blocks completely the stimulatory effects of endogenous or exogenous oestrogens and produces a castration-like involution of the uterus without affecting the hypothalamic-pituitary-ovarian axis. If analogous effects were achieved in patients, peripherally-selective complete oestrogen withdrawal would occur, which presents a novel pharmacological option not achieved by any current treatment. Studies with human breast cancer cells showed that ICI 164,384 reduced to a greater extent than did tamoxifen, the mitotic fraction. This difference may reflect a synergistic stimulatory interaction between serum growth factors like insulin, and the partial agonist effect of tamoxifen which is not seen with ICI 164,384. In long-term culture in the presence of ICI 164,384 no resistant cell lines developed, as has been observed previously in studies with tamoxifen. Pure antioestrogens might thus have a further therapeutic advantage over partial agonists like tamoxifen in reducing the probability of treatment failure due to the regrowth of tumours from resistant cells.

Antineoplastic Agents↗
PubMed1990

A comparative study of the interaction of oestradiol and the steroidal pure antioestrogen, ICI 164,384, with the molybdate-stabilized oestrogen receptor.

The kinetics of binding of oestradiol and the steroidal pure antioestrogen ICI 164,384 to the molybdate-stabilized oestrogen receptor, partially purified from pig and human uterine tissue, were determined. ICI 164,384 bound directly to the oestrogen receptor protein and the kinetic parameters of this interaction were, in general, similar to those for the binding of oestradiol, regardless of the source of the receptor protein. However, the rate of association of oestradiol, regardless of the source of the receptor protein. However, the rate of association of the antagonist with the receptor protein was slower when compared to that of oestradiol. Furthermore, the concentration of binding sites for the two ligands was of the same order. The binding of oestradiol resulted in a steroid-receptor complex which could be transformed in vitro, to a form with increased affinity for DNA-cellulose. However, the complex formed between ICI 164,384 and the receptor protein did not show increased affinity for DNA-cellulose when exposed to conditions that transformed agonist-receptor complexes. Therefore, the binding of ICI 164,384 to the oestrogen receptor protein results in a suppression of the transformation process. A similar suppression in vivo may account for the pure antagonist properties of ICI 164,384.

Animals↗