Points of Action of Estrogen Antagonists and a Calmodulin Antagonist Within the MCF-7 Human Breast Cancer Cell Cycle1

(CANCER RESEARCH 49, 2398-2404, May 1, 1989] Points of Action of Estrogen Antagonists and a Calmodulin Antagonist within the MCF-7 Human Breast Cancer Cell Cycle1

Elizabeth A. Musgrove,2 Alan E. Wakeling, and Robert L. Sutherland3 Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, NSW, 2010, Australia IE. A.M., R.L.S.J; and the Imperial Chemical Industries PLC, Pharmaceuticals Division, Macclesfield, Cheshire SK10 4TG, United Kingdom [A. E. W.Â¡

ABSTRACT potent antitumor activity. Because of this property and the low incidence of side-effects when administered to patients, one of Tamoxifen and other structurally related nonsteroidal antiestrogens these agents, tamoxifen, has become the treatment of choice possess properties in addition to their estrogen antagonist activity includ for hormone-responsive human breast cancer (1-5). ing inhibition of both calmodulin and protein kinase C. The present studies were designed to test whether the estrogen-reversible (estrogen The molecular basis of the antitumor activity of tamoxifen receptor mediated) and estrogen-irreversible effects of nonsteroidal anti- has not been fully elucidated but has been the subject of exten estrogens on cell cycle progression in vitro were mediated at the same or sive study especially in human breast cancer cell lines in culture. different points within the cell cycle and if the estrogen-irreversible effects Early studies revealed that the effects of tamoxifen on breast coincided temporally with that of a calmodulin antagonist, R24571. cancer cell proliferation were confined to ER4-positive cells and Initial experiments investigated the effects of ICI 164384, a pure that these effects could be reversed by the simultaneous or estrogen antagonist, on proliferation kinetics in asynchronous cultures of subsequent addition of estradiol to the culture medium (6-8). MCF-7 human breast cancer cells. At concentrations > l UM ICI 164384 These observations led to the general belief that tamoxifen significantly reduced growth rate while at >50 nM, ICI 164384 completely acted simply as an estrogen antagonist with competitive inhi arrested growth after the first 24 h of exposure. Concentrations up to 5 bition at the level of the ER. More extensive studies in this UM failed either to cause more profound effects on growth or induce cytotoxicity. Growth inhibition was associated with a decrease in the laboratory revealed that tamoxifen and its metabolites had both proportion of S phase cells and an accumulation of cells in d phase, and estrogen-reversible and estrogen-irreversible effects on prolif was completely reversed by the simultaneous addition of equimolar eration in ER-positive breast cancer cell lines. These effects estradiol. were accompanied by distinctive changes in cell cycle kinetic In order to identify the points of action within the cell cycle of ICI parameters and were, in turn, different from the observed effect 164384, and the estrogen-reversible and estrogen-irreversible compo of high drug concentrations on inhibition of proliferation in nents of the nonsteroidal estrogen antagonist, hydroxyclomiphene, and ER-negative cell lines (9-14). Such data raised the possibility the calmodulin antagonist, R24571, experiments were undertaken with that at least in vitro, tamoxifen had antiproliferative activity MCF-7 cells synchronized by mitotic selection. The mean point of action additional to that attributable to its estrogen antagonist prop was assessed by delaying addition of the drugs for increasing time periods following mitotic selection and using DNA flow cytometry to determine erties. Although several other nonsteroidal antiestrogens shared the proportion of the population affected by drug administration at a these properties of tamoxifen in vitro (15-18) the contribution, specific time within d phase. These studies showed that sensitivity to if any, of mechanisms other than estrogen antagonism to the ICI 164384 was restricted to the early part of (,, phase and that the antitumor effects of tamoxifen in vivo has yet to be assessed. mean time of action was 4.9 h after the beginning of d for this pure Attempts to address this issue both in vitro and in vivo have estrogen antagonist. The mean times of action of the estrogen-reversible been hampered by the lack of molecules with pure antiestro- (4.1 h into d phase) and estrogen-irreversible (4.1 h) mechanisms of genic activity. However, in 1987 a new series of steroidal action of hydroxyclomiphene, and R24S71 (4.0 h), all appeared to be antiestrogenic molecules was described, which, unlike the non within a similar time frame in early to mid <., phase. steroidal counterparts, failed to demonstrate estrogen agonist It is concluded that ICI 164384 inhibits breast cancer cell proliferation activity in a wide variety of experimental systems (19-21). by inducing a transition delay in GÃ¬phase and that the point of action of this pure estrogen antagonist in early GÃ¬phase is indistinguishable These unique compounds have provided the tools necessary to temporally from that of nonsteroidal antiestrogens and calmodulin antag distinguish the antiproliferative properties of tamoxifen that onists. are due to estrogen antagonism from those that are independent of its estrogen antagonism. Some in vitro experiments aimed at distinguishing these effects are reported here. INTRODUCTION Tamoxifen is now known to bind with relatively high affinity The nonsteroidal antiestrogens, of which tamoxifen is the to a number of intracellular proteins in addition to the ER. most studied molecule, are a group of derivatives of the syn These include: a specific high affinity antiestrogen binding site thetic estrogen, triphenylethylene. These molecules display a (22-24), calmodulin (25), protein kinase C (26), cytochrome P- diversity of biological properties and behave as estrogen ago 450 (27), and muscarinic- (28), dopamine- (29), and histamine- nists, estrogen antagonists, or partial agonists/partial antago receptors (30). Of these potential mediators of tamoxifen action nists depending upon the species, tissue or response parameter independent of the ER, calmodulin antagonism was chosen for under study. In a number of hormone-responsive tumors, par investigation in this study. Calmodulin antagonists have been ticularly breast carcinoma, nonsteroidal antiestrogens possess shown to inhibit proliferation of a variety of cell types (31-34) including breast cancer cells (35, 36). The growth arrest has Received 12/1/88; accepted 2/6/89. The costs of publication of this article were defrayed in part by the payment been attributed to sites of action in early d (33, 37) and of page charges. This article must therefore be hereby marked advertisement in blockade near the d/S boundary (32, 37). Furthermore, pre- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Supported by the National Health and Medical Research Council of Australia 4The abbreviations and trivial names used are: ER, estrogen receptor; ICI and MLC-Life Ltd. 164384, iV-n-butyl-A'-methyl-11 -[3,170-dihydroxyestra-1,3,5( 10)-trien-7a-yl]un- 2 PhD scholar supported by the Government Employees Assistance to Medical decanamide: hydroxyclomiphene, 1-(4-/3-diethylaminoethoxyphenyl)-1 -(4-hydr- Research Fund. oxyphenyl)-2-chloro-2-phenylethylene; R24571, calmidazolium {1-[bis(p-chloro- ' To whom requests for reprints should be addressed at the Garvan Institute phenyl)methyll-3-[2,4-dichloro-/3-(2,4)dichlorobenzyloxy]phenethyl]imidazoli- of Medical Research, St. Vincent's Hospital, Sydney, NSW, 2010, Australia. nium chlor idei; Ej, estradiol 170. 2398

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1989 American Association for Cancer Research. CELL CYCLE EFFECTS OF ESTROGEN ANTAGONISTS liminary experiments from this laboratory5 (38) showed that 150 flasks set up at 1 x IO6cells/flask, yielded sufficient cells to set up the inhibition of breast cancer cell proliferation in vitro by two four to five T-25 flasks (i.e., 1-2 x 10" cells/T-150) and took aproxi- mately 30 min. The time at which the flasks were placed into the phenothiazine calmodulin antagonists was accompanied by incubator at 37Â°Cfollowing mitotic selection was designated 0 h and changes in cell cycle kinetic parameters qualitatively similar to those seen with nonsteroidal antiestrogens, i.e., accumulation taken to be the commencement of GÃ¬sinceDNA analysis demonstrated that almost all of the selected population had completed mitosis and of G i phase cells at the expense of S phase cells. These data, entered d at that time (11). taken together with the correlation between calmodulin antag Drug Treatment. ICI 164384, hydroxyclomiphene, R24571, and E2 onism and estrogen-irreversible growth inhibitory potency in a were stored at -20Â°C as 1,000-fold concentrated stock solutions dis series of agents including both triphenylethylene antiestrogens solved in analytical reagent grade ethanol or AVV-dimethylformamide and calmodulin antagonists demonstrated using MCF-7 cells and added directly to the culture medium so that the final vehicle (36), provide support for the hypothesis that some of the cell concentration was no greater than 0.2%. Control flasks received vehicle cycle changes induced by antiestrogens may be attributable to alone to the same final concentration and this was shown to have no calmodulin antagonism. significant effect on MCF-7 proliferation when compared with control Previously published experiments aimed at identifying the cultures without vehicle. point of action of antiestrogens within the cell cycle suffer from In general synchronized cells were exposed to each compound for a period commencing at the indicated time after mitotic selection and a number of limitations. Studies with synchronous populations continuing until harvest for DNA analysis at 16 h. In some experiments, of MCF-7 cells showed that tamoxifen inhibits cell cycle pro which involved treatment with 7.5 Â¿IMhydroxyclomiphene in 3-h pulses, gression within a distinct time-frame in early to mid GÃ¬phase the culture medium was removed at the end of the exposure time and (11). However, because these experiments were conducted with the monolayer washed gently with two aliquots of 2 ml warm fresh micromolar concentrations of tamoxifen it is unclear whether medium before refeeding with 5 ml warm fresh culture medium. The the effects observed at the highest concentrations were due cells were then returned to the incubator at 37Â°Cand harvested 16 h entirely to the estrogen antagonist properties of tamoxifen. The after mitotic selection. only other study (39) employed the semiquantitative technique DNA Analysis. Harvested cells were collected by centrifugation (350 x g x 5 min), drained, and then stained by resuspension at a concen of chromosome condensation to document an effect in early G, tration of no more than 2 x IO5cells/ml in culture medium to which phase. The availability of ICI 164384 has allowed precise iden ethidium bromide (40^g/ml), mithramycin (12.5 ^g/ml), Triton X-100 tification of effects on MCF-7 breast cancer cell cycle progres (0.2% v/v), and MgCl2 (7.5 mM) had been added (42). The samples sion due to estrogen antagonism and facilitated a comparison were stored at 4Â°Cfor later flow cytometric DNA analysis, which was with the estrogen-irreversible effects of nonsteroidal antiestro performed using a FACStar instrument and accompanying DNA analy gens and those of calmodulin antagonism. sis software (Becton Dickinson Immunocytochemistry Systems, Moun tain View, CA). The DNA stained cells were illuminated with laser MATERIALS AND METHODS light at 457 nm and the resulting emission from the DNA fluoro- chromes measured at 585 nm (585/42 bandpass filter). The cell cycle Reagents. Insulin was purchased from CSL-Novo, Parramatta, Aus phase fractions were estimated using a model in which a second-order tralia; other tissue culture materials were obtained from Flow Labora polynomial is fitted to S phase (SFIT), or for histograms of synchro tories, Sydney, Australia. ICI 164384 (19-21) was from ICI Pharma nized populations, using an algorithm which iteratively fits the S phase ceuticals Division, Macclesfield, UK. Hydroxyclomiphene was synthe- region of the DNA histogram with the sum of a number of broadened sised as previously described (40) and donated by Dr. P. Ruenitz, rectangles [SOBR (43)]; in the second model in addition to the phase College of Pharmacy, University of Georgia, Athens, GA. Other re fractions the standard deviation of the estimation of each phase is agents with the exception of mithramycin (Pfizer, Sydney, Australia) computed and this was used to indicate the error in the phase fractions. were purchased from the Sigma Chemical Co. (St. Louis, MO). Data Analysis. Where data points were obtained by subtraction of Tissue Culture. Stock cultures of MCF-7 cells, originally obtained the %Gi of a control histogram from that of a treated sample, the error from Dr. C. McGrath, Meyer L. Prentis Cancer Center, Detroit, MI, in the difference was calculated as the square root of the sum of the in their 299th passage, were maintained as previously described (10) in standard deviation of the control %Gi estimate, squared, and the RPMI 1640 medium containing 0.06% phenol red [a known estrogenic standard deviation of the treated %Gi estimate, squared. Where the compound with growth stimulatory activity for these cells (41)] and data from several experiments are pooled, the error displayed is the supplemented with 10% fetal calf serum, 6 ITIML-glutamine and 10 n%/ square root of the sum of the standard error of the mean of the pooled ml porcine insulin. Under these conditions MCF-7 cells grow maxi data, squared, and the mean errors in estimation of each individual mally and are ideally suited for studies on growth inhibitors. data point, squared. The time of action of a particular agent within the Growth inhibition of asynchronous cultures was measured by plating cell cycle was calculated as the time corresponding to inhibition 50% 5 x IO4cells in exponential growth phase into T-25 tissue culture flasks ofthat observed when drug treatment began l h after mitotic selection, in 5 ml of the same medium used for stock cultures but with the using the equation of a line of best fit determined by linear regression. concentration of fetal calf serum reduced to 5%. The following day, when the cell number had approximately doubled, either ICI 164384, E2 or both were added as described below. After approximately four RESULTS population doublings of the control cultures, i.e., 5-6 days, or more frequently, triplicate flasks were harvested with 0.05% trypsin-0.02% Growth Inhibition of Asynchronous Cells with ICI 164384 or EDTA in Ca2+-Mg2+-freephosphate buffered saline (1.5 mM KH2PO4- Hydroxyclomiphene. In order to investigate the effects of the 8.1 mM Na2HPO4-2.7 mM KC1-140 mM NaCI). Viable cells were pure estrogen antagonist, ICI 164384, on MCF-7 cell prolifer counted using a hemacytometer under phase-contrast microscopy. The ation kinetics exponentially growing cultures were treated with cells from each treatment group were then pooled and stained for later different concentrations of the drug for up to 6 days. At con DNA analysis. centrations between 1 and 50 nivi, ICI 164384 inhibited the Synchronization by mitotic selection was achieved as previously proliferation of MCF-7 cells in a dose-dependent fashion after described (11). Mitotic cells were harvested at 4-h intervals from Day an approximate doubling of cell numbers during the first 24 h 2 or 3 cultures and plated into T-25 flasks in 5 ml fresh warm medium (Fig. 1). Concentrations up to 5 Â¿Â¿Mwereno more effective than made as above but with 5% fetal calf serum. Each harvest, from 16 T- 50 nM, which was cytostatic after the first 24 h of exposure. 5C. K. W. Watts, E. A. Musgrove, and R. L. Sutherland, unpublished obser There was evidence of cytotoxicity only at concentrations >5 vations. MM,where cell numbers were below the drugging density. The 2399

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8 1 2 1 6 .1 1 10 1001 000 10000 IC1164384 concentration (nM) 4.0 Time after mitotic selection (h) Fig. 2. Experimental design used to determine time of action. MCF-7 cells were synchronized by mitotic selection and returned to 37'C at time 0. Parallel Time (days) cultures were treated with growth inhibitory agents at a series of successively later times and all flasks were harvested at Id h. D. drug exposure. For these cells the Fig. 1. Effect of ICI 164384 on the growth of MCF-7 human breast cancer cells. Exponentially growing cells (5 x HI4)were plated into T-2S tissue culture mean length of d phase is 9 h. flasks in S ml of RPMI 1640 medium supplemented with 5% fetal calf serum, and treatment with ICI 164384 commenced the following day (Day 0). Cells were proportion of the population will be susceptible to its action if subsequently harvested and viable cells counted under phase contrast microscopy. Treatments shown are: control (â€¢).I (D). 5 (A), 10 (A), and 100 IÂ»)nM ICI the delay is sufficient to allow some cells to progress past the 164384. The data are the means of a representative experiment performed in last sensitive point. Those cells which, by virtue of their position triplicate, in which the SE was less than 7.3%. Inset, cell growth inhibition by ICI later in d, are no longer sensitive, will continue into S phase. 164384 (â€¢)andhydroxyclomiphene (D), expressed as a percentage of the control cell number at Day S or 6, i.e.. after four population doublings of the control If the population is then harvested at some later time which cells. Data points for ICI 164384 represent the mean of three to nine flasks from allows distinction to be made between those cells which have up to three experiments, the results of which varied by no more than 16%, while progressed into S phase and those retained in (;,, it is possible the data for hydroxyclomiphene are redrawn from refs. 16 and 17. to determine the relative proportion of sensitive cells at various times within (Â¡,.Half maximal inhibition would be expected to reduction in growth rate was preceded by a marked reduction occur when the mean age of the cells at the time drug treatment in the proportion of cells in S and G2 + M phases and concom began was equal to the time of action, i.e., when half the itant increase in the d fraction (data not shown). These changes population was before, and half the population after, the sen in cell cycle kinetic parameters were apparent after 24 h, max sitive time. imal by 3 days (at which time decreases in %S phase of up to Control cultures harvested 1-24 h after mitotic selection 70-80% were observed), and maintained over a further 3-day demonstrated a high degree of synchrony, in that the ( Â¡,fraction exposure. Concentrations of 10 or 100 nM ICI 164384 admin was initially 80-90% and after 6-8 h fell to a minimum of 25- istered in the simultaneous presence of equimolar E2 were 30% (data not shown). The mean time to exit d for control ineffective in reducing either the growth rate or percentage of cells set up in parallel with the treated cultures was 8.7 Â±0.3 h cells in S phase (data not shown). (mean Â±SE, N = 10). On the basis of these experiments 16 h Hydroxyclomiphene has qualitatively similar effects on cell after mitotic selection was chosen as an appropriate time to growth and phase distribution when tested under the same determine the number of cells retained in d following drug experimental conditions (Fig. 1, inset). The ability of estrogen treatment, since at that time cells initiating DNA synthesis had to reverse the growth inhibition differs in the two portions of progressed into late S phase (enabling good discrimination the hiphasic dose-response curve: at concentrations <1 /Â¿M between sensitive and insensitive cells) but had not completed hydroxyclomiphene effects are wholly reversed by the simulta mitosis (Fig. 3 and rÃ©f.11).DNA histograms of cells exposed neous addition of E2, while at higher concentrations they are to 7.5 fiM hydroxyclomiphene following delays of up to 6 h incompletely reversed (17). Thus at a concentration of 7.5 /Â¿M, after, and harvested 16 h after, mitotic selection are shown in used in the experiments with synchronized cells, hydroxyclom Fig. 3. Maximal inhibition of progression into S phase occurred iphene would exert both the E2-reversible and the E2-irreversible when drug treatment began less than 2 h after the beginning of components of growth inhibition of nonsteroidal antiestrogens. d. When treatment was commenced thereafter, decreasing Determination of Times of Action within the Cell Cycle. We numbers of cells were affected until after a 6 h delay very few next undertook experiments designed to identify more precisely cells were retained in GÃ¬.Atthis concentration, hydroxyclom the point of action within GÃ¬phase of ICI 164384, and of iphene caused a slight delay in transit through S phase, in hydroxyclomiphene, and the cai modulili antagonist R24S71. addition to the primary effect of arrest in GÃ¬,asevidenced by The experimental design illustrated in Fig. 2 was based on the the lower DNA content of the cohort of cells in S phase following reasoning: if the exposure of synchronized cells to a compared with control histograms (Fig. 3). compound which exerts its effects at a specific point in d is The reduction in sensitivity to hydroxyclomiphene apparent delayed until the cells have completed part of d, a decreasing with delayed commencement of drug treatment within d oc- 2400

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Fig. 3. DNA histograms of cells treated with 7.5 JIMhydroxyclomiphene. The experimental design was as described in Fig. 2. Each histogram contains data from 16,000 to 19,000 cells and has a coefficient of variation between 3.1 and 3.6%. curred as the duration of exposure to the drug was reduced from 16 to 10 h. However it is not simply due to decreased total exposure time. Fig. 4 presents the results of experiments in which 7.5 tiM hydroxyclomiphene was administered as a 3-h pulse beginning 0, 3, or 6 h after mitotic selection. Again the sensitivity of the cells was reduced as they moved through GÃ¬, with the pulse commencing 6 h after selection having a signif icantly smaller effect (P < 0.025) than that commencing at 0 h. This indicates that cells in late GÃ¬areindeed relatively insen sitive to 7.5 ÃÃMhydroxyclomiphene. Time after mitotic selection (h) We then sought to determine the precise time of action within Fig. 5. Effect of the time of exposure to hydroxyclomiphene on the proportion GÃ¬bymaking observations 1 h apart, up to a maximum of 8 h of synchronized MCF-7 cells remaining in GÃ¬phase.The experimental design is after mitotic selection. The number of cells blocked in GÃ¬was as described in Fig. 2, using 7.5 nM hydroxyclomiphene alone (â€¢)or in the presence of 1 tÂ¡ME2(D). Points, mean of two to four individual experiments in obtained by subtraction of the %G\ of parallel control samples which the %G\ of parallel control samples has been subtracted from each data from the %Gi of the drug-treated sample. Fig. 5 shows the point; bars, combination of errors in data points and the SE of their mean, calculated as described in "Materials and Methods." Regression lines are calcu reduction in numbers of such cells with increasing delay of lated using data from 1 to 6 h inclusive; r' = 0.99 for both hydroxyclomiphene administration of hydroxyclomiphene in the presence or ab alone and hydroxyclomiphene plus E2. sence of 1 nM E2. Under both sets of experimental conditions half-maximal retention of cells in GÃ¬occurred at 4.1 h, estab Although the effect is more modest than that achieved with 7.5 lishing the temporal identity of the E2-reversible and E2-irre- /Â¿Mhydroxyclomiphene it can be concluded firstly that the versible actions of this drug. maximum effect is seen only for cells early in GÃ¬whilelate in Similar experiments were performed using 100 nM ICI GÃ¬most cells are insensitive to ICI 164384, and secondly that 164384. In the presence of equimolar E2, ICI 164384, failed to under these experimental conditions the growth arrest induced increase the %G\ compared to untreated control cells (Fig. 6). by this compound is abrogated by the simultaneous addition of 2401 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1989 American Association for Cancer Research. CELL CYCLE EFFECTS OF ESTROGEN ANTAGONISTS

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2468 1 0 2468 Time after mitotic selection (h) Time after mitotic selection (h) Fig. 7. Effect of the time of exposure to the calmodulin antagonist R24S71 Fig. 6. Effect of the time of exposure to ICI 164384 on the proportion of on the proportion of synchronized MCF-7 cells remaining in G, phase. R24571 synchronized MCF 7 cells remaining in GÃ¬phase.Cells were exposed to 100 nM was administered at a concentration of 2.5 JIMas described in Fig. 2. Points, mean ICI 164384 in the presence (D) or absence (â€¢)ofequimolar E2, commencing at of two to three experiments in which the %G, of parallel control samples has the indicated time after mitotic selection, according to the protocol illustrated in been subtracted from each data point; bars, errors calculated as described in Fig. 2. Points, means of data from two or three separate experiments. The %d "Materials and Methods." Data from 1 to 6 h have been used to calculate the line of parallel control samples has been subtracted; error bars, a combination of the of best fit (r2 = 0.98). errors in the estimation of individual data points as described in "Materials and Methods." The line of best fit was calculated by linear regression analysis of data between 1 and 6 h inclusive; r2 = 1.00. The present study confirms earlier reports (19, 20) that the pure estrogen antagonist ICI 164384 inhibits the proliferation equimolar E2. The half-maximal inhibition of initiation of DNA of human breast cancer cells in culture. The observed pattern synthesis occurred 4.9 h after the beginning of G|. of cell cycle kinetic effects is similar to that seen with the To test the hypothesis that the calmodulin antagonist activity nonsteroidal antiestrogens and their hydroxylated derivatives of tamoxifen provides a potential mechanism for mediation of (compare refs. 12, 14, 16, 17, 39, 44, 45) in inhibiting ER- the E2-irreversible and therefore putatively non-ER-mediated positive breast cancer cell growth and is consistent with a site actions of nonsteroidal, triarylethylene antiestrogens, the time of action early in GÃ¬.Ifthis is the major site of action and the of action of the potent calmodulin antagonist, R24571, was remainder of the cell cycle is relatively unaffected, the majority determined using the same protocol. A concentration of 2.5 MM of the cell population will be capable of progression through was chosen on the basis of previous experiments using asyn the remainder of d, S, ( ;.â€¢.andmitosis and back into GÃ¬phase chronous cultures, which showed a degree of growth inhibition in the first cell cycle of exposure. This would account for the similar to that of 7.5 nM hydroclomiphene, and concomitant increases in %G\ phase cells.5 The results are presented in Fig. initial increase in cell number at control rates during the first 24 h of drug exposure as seen in Fig. 1. However, few of the 7 where again sensitivity to the agent declined with transit daughter cells will commence DNA synthesis because of their through GÃ¬,and the half maximal effect occurred at 4.0 h. inability to overcome the block in early GÃ¬,resulting in a sharp DNA histograms of R24571-treated cells showed that this drop in %S phase and in population doubling time. At concen concentration of R24571 did not produce evidence of a second trations which are cytostatic but not cytotoxic this mechanism block in early S phase (data not shown), as has been observed is entirely estrogen-reversible and therefore presumably me with some calmodulin antagonists in other cell types (37). diated solely through the ER. Although the cell cycle correlates of the growth arrest, i.e., increase in the number of cells in GI DISCUSSION phase and decrease in %S phase, are qualitatively similar to those observed using other, nonsteroidal, estrogen antagonists, There is accumulating evidence to suggest that nonsteroidal and ICI 164384 is growth inhibitory over a concentration range antiestrogens have both estrogen-reversible and estrogen-irre (1-100 HM) similar to the estrogen-reversible actions of vinyl versible, but cell cycle specific, components to their action as substituted hydroxytriphenylethylenes, e.g., hydroxyclomi- growth inhibitory agents in vitro (9-18, 36, 38). Detailed cell phene (16, 17), these effects are quantitatively greater for ICI kinetic studies employing synchronized cells have been re 164384. This is illustrated by the observation that in the pres stricted to concentrations of tamoxifen in the micromolar range ence of 100 nM ICI 164384 the cell number is reduced by 88% (11). The possibility that at these high concentrations the compared to control, after four doublings of the untreated predominant effect is not mediated through the ER prompted control population, whereas nonsteroidal antiestrogens with an examination of the kinetics of cells exposed to a pure high affinity for the ER such as hydroxyclomiphene have a estrogen antagonist, specifically to determine its point of action maximum entirely E2-reversible growth inhibition of 60-70% within the cell cycle and to establish the relationship between (see inset to Fig. 1). It is possible that the estrogen agonist this and the observed effects of nonsteroidal estrogen antago activity of nonsteroidal antiestrogens demonstrated both in vivo nists. (1-5), and in vitro in estrogen-free culture conditions (46, 47), 2402

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1989 American Association for Cancer Research. CELL CYCLE EFFECTS OF ESTROGEN ANTAGONISTS is also operating under the experimental conditions described from a nonbinding to a hormone-binding state (51). If, as here and reduces the apparent efficacy of the growth inhibitory suggested by the authors (50, 51), this process is critical to action. estrogen action the parallels between inhibition of receptor The experiments designed to identify the point of action of phosphorylation by calmodulin antagonists and antiestrogens antiestrogens within the cell cycle show that the last point at are obvious and would provide a potential explanation for the which ICI 164384 is effective is located 4-5 h after the begin coincidence of the effects of these agents on breast cancer cell ning of GÃ¬,around 4 h before initiation of DNA synthesis. This cycle progression reported here. antiestrogen-sensitive step is coincident with both the E2-reversible and E2-irreversible actions of hydroxyclomiphene. Pre REFERENCES viously published data had placed tamoxifen sensitivity within a 2 h window in early-to-mid d (11), and indeed data obtained 1. Sutherland, R. L., and Jordan, V. C. Non-steroidal Antiestrogens: Molecular using 7.5 fiM tamoxifen and an identical experimental design Pharmacology and Antitumour Activity. Sydney: Academic Press, 1981. to that detailed here6 are in close agreement with the timing 2. Sutherland, R. L., and Murphy, L. C. Mechanisms of oestrogen antagonism by nonsteroidal antioestrogens. Mol. Cell. Endocrino!., 25: 5-23, 1981. observed in the present experiments. 3. Furr, B. J. A., and Jordan, V. C. The pharmacology and clinical uses of tamoxifen. Pharmacol. & Then, 25: 127-205, 1984. Although the blockade in GÃ¬observed during the first cell 4. Jordan, V. C. 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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1989 American Association for Cancer Research. Points of Action of Estrogen Antagonists and a Calmodulin Antagonist within the MCF-7 Human Breast Cancer Cell Cycle

Elizabeth A. Musgrove, Alan E. Wakeling and Robert L. Sutherland

Cancer Res 1989;49:2398-2404.

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