Points of action of estrogen antagonists and a calmodulin antagonist within the MCF-7 human breast cancer cell cycle.
Tamoxifen and other structurally related nonsteroidal antiestrogens possess properties in addition to their estrogen antagonist activity including inhibition of both calmodulin and protein kinase C. The present studies were designed to test whether the estrogen-reversible (estrogen receptor mediated) and estrogen-irreversible effects of nonsteroidal antiestrogens on cell cycle progression in vitro were mediated at the same or different points within the cell cycle and if the estrogen-irreversible effects coincided temporally with that of a calmodulin antagonist, R24571. Initial experiments investigated the effects of ICI 164384, a pure estrogen antagonist, on proliferation kinetics in asynchronous cultures of MCF-7 human breast cancer cells. At concentrations greater than 1 nM ICI 164384 significantly reduced growth rate while at greater than or equal to 50 nM, ICI 164384 completely arrested growth after the first 24 h of exposure. Concentrations up to 5 microM failed either to cause more profound effects on growth or induce cytotoxicity. Growth inhibition was associated with a decrease in the proportion of S phase cells and an accumulation of cells in G1 phase, and was completely reversed by the simultaneous addition of equimolar estradiol. In order to identify the points of action within the cell cycle of ICI 164384, and the estrogen-reversible and estrogen-irreversible components of the nonsteroidal estrogen antagonist, hydroxyclomiphene, and the calmodulin antagonist, R24571, experiments were undertaken with MCF-7 cells synchronized by mitotic selection. The mean point of action was assessed by delaying addition of the drugs for increasing time periods following mitotic selection and using DNA flow cytometry to determine the proportion of the population affected by drug administration at a specific time within G1 phase. These studies showed that sensitivity to ICI 164384 was restricted to the early part of G1 phase and that the mean time of action was 4.9 h after the beginning of G1 for this pure estrogen antagonist. The mean times of action of the estrogen-reversible (4.1 h into G1 phase) and estrogen-irreversible (4.1 h) mechanisms of action of hydroxyclomiphene, and R24571 (4.0 h), all appeared to be within a similar time frame in early to mid G1 phase. It is concluded that ICI 164384 inhibits breast cancer cell proliferation by inducing a transition delay in G1 phase and that the point of action of this pure estrogen antagonist in early G1 phase is indistinguishable temporally from that of nonsteroidal antiestrogens and calmodulin antagonists.
Musgrove
E A
EA
Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, NSW, Australia.
Wakeling
A E
AE
Sutherland
R L
RL
D001943:Breast Neoplasms / Q000473:pathology*; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D002453:Cell Cycle / Q000187:drug effects*; D002996:Clomiphene / Q000031:analogs & derivatives / Q000494:pharmacology; D004247:DNA / Q000032:analysis; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology*; D005260:Female; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D053284:Polyunsaturated Alkamides; D014407:Tumor Cells, Cultured
D002147:Calmodulin; D004965:Estrogen Antagonists; D007093:Imidazoles; D053284:Polyunsaturated Alkamides; D002996:Clomiphene; C031938:calmidazolium; D004958:Estradiol; C050622:4-hydroxyclomiphene; C051781:ICI 164384; D004247:DNA