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[CANCER RESEARCH 43. 3583-3585, August 1983] Effects of Tamoxifen on Human Breast Cancer Cell Cycle Kinetics: Accumulation of Cells in Early GìPhase1 C. Kent Osborne,2 David H. Boldt, Gary M. Clark, and Jeffrey M. Trent3 Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78284 [C. K. O., D. H. B., G. M. C.], and the University of Arizona Health Science Center, Tucson, Arizona 85724 [J. M. T.] ABSTRACT We have studied the effects of tamoxifen on the cell cycle kinetics of the endocrine-responsive MCF-7 human breast cancer cells. Tamoxifen inhibits proliferation of MCF-7 cells. The tritiated thymidine labeling index is markedly reduced by tamoxifen, in dicating a reduction in the fraction of cells in S phase. Flow cytometry of mithramycin-stained cells reveals that cells accu mulate in Gìphase, with a concomitant depletion of S- and G2M-phase cells with tamoxifen. Mapping of G1-phase cells by morphology of prematurely condensed chromosomes demon strated that tamoxifen-treated cells accumulate in early G,. These studies indicate that tamoxifen inhibits proliferation of MCF-7 human breast cancer cells by invoking a transition delay early in the d phase of the cell cycle. INTRODUCTION The antiestrogen tamoxifen is now widely used for the treat ment of patients with breast cancer. However, its mechanism of action is not totally defined. Although specific binding sites for tamoxifen have been identified recently in certain tissues (15), tamoxifen, like other antiestrogens, is thought to exert its effects by binding to estrogen receptor and then translocating it to the nucleus, where steps leading to estrogen-regulated events such as tumor growth are inhibited (6). Tamoxifen has been shown to inhibit breast tumor growth in experimental animals (8) and to inhibit a variety of biochemical events in cultured human breast cancer cells including nuclear processing of estrogen receptor (7), the activity of key enzymes such as DNA polymerase (3), and cell proliferation (10). Little is known about the effects of tamoxifen on cell cycle kinetics. Previous studies using [3H]thymidine incorporation techniques suggest that tamoxifen may block cells in a uniform phase of the cell cycle (9), possibly in G, (12, 16, 17). Here, we demonstrate that tamoxifen causes a transition delay early in the d phase of the cell cycle. MATERIALS AND METHODS Cell Proliferation. The tissue culture methodology used for propaga tion of the MCF-7 human breast cancer cells has been described previ ously (14). For determination of the effects of tamoxifen on cell prolifer ation, MCF-7 cells were replicately plated into multimeli culture dishes in tissue culture medium (Richter's Improved Eagle's Minimal Essential Medium ZO; Grand Island Biological Co., Grand Island, N. Y.) supple mented with insulin (1.0 HM) and 5% bovine serum. After 24 hr, plating medium was replaced with medium supplemented with 5% dextrancoated charcoal-stripped bovine serum to reduce endogenous estro' This work was supported by NIH Grant CA 30251 and by an institutional grant from the American Cancer Society (IN-116C). 1 To whom requests for reprints should be addressed. 3 Recipient of National Cancer Institute Grants CA 29476 and CA 23074. Received December 9,1982; accepted May 4,1983. AUGUST gens. After an additional 24 hr, tamoxifen (1 »M)or ethanol (final concen tration, 0.1%) was added to the dishes. At the indicated times, cells were suspended and counted in a hemocytometer. Cell Kinetic Studies. Cells were plated and cultured as described above. At the indicated times, cells were pulsed with tritiated thymidine (0.3 fiCi/ml) and washed twice with PBS,4 and a cell suspension was prepared. Uhe TLI was determined autoradiographically as described previously (11, 14). The cell cycle distribution of MCF-7 cells was determined by flow cytometry of mithramycin-stained cells (1). Cells were washed once and, then, suspended in 0.02% EDTA in PBS. After a 5to 10-min incubation, a single-cell suspension was prepared mechanically by passing the cells through progressively smaller gauge needles (22, 25, and 27 gauge). The cells were pelleted by centrifugaron (800 rpm for 2 min) and resuspended in 0.4 ml of PBS. The cells were then fixed by adding ethanol (final concentration, 70%) while vortexing and stored at 4°.Fixed cells were pelleted by centrifugation, and the ethanol was discarded. Cells were washed once with PBS and, then, resuspended in staining solution (1.0 mg mithramycin:0.87 g NaCI:0.15 ml 1 M MgCb:9.85 ml distilled H2O) to a concentration of 5 x 105 cells/ml. The cells were protected from light at 4°overnight prior to flow cytometry. DNA histo grams were obtained by analyzing a total of 1 x 104 cells on a Coulter Model TPS-1 cell sorter equipped with a 2-watt argon-ion laser which was tuned to 457.9 nM. Histograms were analyzed by the computerassisted method of Dean and Jett (2). The coefficient of variation of the d peak varied from 4 to 8% using this method. Distribution of GìCells. MCF-7 cells were cultured as described above and then incubated with either ethanol (controls) or tamoxifen (1 ¿¿M) for 72 hr. G, cells were mapped by the method of Hittelman and coworkers (4, 5, 18), which analyzes the morphology of PCC. Briefly, mitotic HeLa cells were obtained following a 24-hr exposure to thymidine (2.5 rtiM), followed by a 15-hr exposure to colchicine (0.1 MM). This procedure routinely provides a 97% pure population of mitotic cells (as distinguished by light microscopy following acetoorcein staining). Equal numbers of mitotic HeLa cells and MCF-7 cells were mixed in serumfree medium and pelleted (150 x g for 2 min). Cells were resuspended in 0.5 ml of PBS containing 2000 hemagglutinating units of UV-inactivated Sendai virus (a generous gift of Dr. W. Hittelman, Houston, Texas). Cells were then incubated at 4°for 15 min after the addition of 0.05 ml of 20 rnw MgCl2 and Colcemid (5 ng/mi). Samples were warmed to 37°and incubated for an additional 45 min. Cell pellets were obtained by centrif ugation (150 x g, 5 min), and 7 ml of 0.075 M KCI (prewarmed to 37°) were added for 10 min. Cells were then recentrifuged, the supernatant was removed, and 7 ml of fresh cold fixative (3:1 methanol:glacial acetic acid) were added. Air-dried slides were then prepared and stained with 4% Giemsa (Gurr's R-66). G, PCC were graded on a scale of 1 through 6, depending on the degree of condensation. PCC given values of 1 to 3 (highly condensed) were considered to be in early G,, whereas those given values of 4 to 6 (less condensed) were judged to be in late Gì(4). RESULTS When cells from the well-characterized MCF-7 estrogen recep4The abbreviations used are: PBS, phosphate-buffered saline [NaCI (8 g/ Bter):KCI (0.2 g/liter):Na2HPO4 (1.15 g/liter):KH2PO, (0.2 g/liter); TLI, tritiated thy midine labeling index; PCC, prematurely condensed chromosomes. 1983 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1983 American Association for Cancer Research. 3583 C. K. Osborne et al. tor-positive human breast cancer cell line were incubated with tamoxifen (Chart 1), inhibition of cell proliferation similar to that observed in previous reports (9, 17) was observed. In 5% char coal-stripped serum, control cultures proliferated rapidly and had reached confluence by Day 6. Tamoxifen (1 fiM) slowed the rate of proliferation slightly during the first 48 hr. Thereafter, prolifer ation was markedly reduced, cell number nearly plateaued, and the monolayers remained at a subconfluent density. These data suggest that more than one passage through the cell cycle is required for expression of maximal effects of tamoxifen at these concentrations. When MCF-7 cells were incubated with tamoxi fen for longer durations or were incubated with tamoxifen in serum-free medium, cells would begin to detach from the monolayer and appear as floaters. It is important to emphasize, however, that, under the experimental conditions described in Chart 1, cell number did not decline with tamoxifen. These data suggest that tamoxifen slowed proliferation but did not have an immediate cytocidal effect on the MCF-7 cells. We next examined the effect of tamoxifen on the distribution of MCF-7 cells in the cell cycle. The TLI was used to estimate the fraction of cells in the S phase of the cell cycle (Chart 2). The TLI of control cells growing in medium supplemented with dextran-coated charcoal-stripped calf serum was 35% at the begin ning of the experiment. The TLI decreased slightly over 96 hr in control cultures, probably reflecting depletion of nutrients from the medium and/or density-dependent growth inhibition as the monolayer neared confluence. Tamoxifen resulted in a marked reduction in the TLI, and by 72 to 96 hr, less than 10% of the cell population was in S phase. To determine the effects of tamoxifen on the distribution of MCF-7 cells in the other cell cycle phases, DMA histograms were obtained by flow cytometry of mithramycin-stained cells (Chart 3). After 72 hr in control medium, 72% of cells were in d as revealed by computer analysis of DNA histograms, 19% were in S phase (similar to the TLI result), and 9% were in G2-M phases. Tamoxifen treatment resulted in a marked accumulation of cells in Gì (92%), concomitant with a depletion of cells from the S and Û2-M compartments. When experiments were carried out for longer time periods (6 days), cells exposed to tamoxifen re mained in Gì,indicating that the block in G, phase was not temporary. It has been reported that serum-deprived or plateau-phase 40 n £ 3020 3 .1 •p.0 E 24 72 48 96 Time (hours) Chart 2. Effect of tamoxifen on the TLI of MCF-7 cells. Control G, = 7I.7% S =19.1 % G2M = 9.2% 0) -Q 128 TAM \fj.M G, = 91.9% S =81% G2M = 0.0% 128 Relative Fluorescence Intensity (Channel Number) Chart 3. Effect of tamoxifen (TAM) on the cell cycle distribution of MCF-7 cells. A, control cells; B, tamoxifen for 72 hr. Hatched area, computer estimate of the Sphase fraction of cells. normal (nontransformed) cells accumulate in early Gì,whereas malignant or transformed cell populations accumulate in late Gì (4). Furthermore, certain metabolic inhibitors, such as actinomycin D or cycloheximide, block cells in early Gìin contrast to inhibitors of DNA synthesis, such as thymidine or hydroxyurea, which block cell cycle progression at the d-S boundary (4). Chart 1. Effect of tamoxifen on proliferation of MCF-7 cells. Cells growing in medium supplemented with 5% dextran-coated charcoal-stripped bovine serum were incubated in the absence (•)or presence (O) of tamoxifen (1 ,,M). Fresh medium and tamoxifen were exchanged for spent medium on Day 4. Points, mean of triplicate determinations; bars, S.D. 3584 Thus, we were interested in determining where in the d phase the tamoxifen block occurred. Utilizing the morphology of PCC, cells can be classified as occurring early or late in the d phase based upon the structural criteria of Hittleman and Rao (4). This is possible because the chromosomes from early d PCC are highly condensed, whereas those from late G, PCC are long and extended. Morphological assessment of PCC (Table 1) revealed that 62% of GìPCC in control cultures were in late Gì.In contrast, only 35% of tamoxifen-treated d PCC were present in late G, (p = 0.001). The majority (65%) of tamoxifen-treated cells had accumulated early in d. Furthermore, exact chromo some counts of d PCC revealed that both control and tamoxi fen-treated d cells had similar chromosome numbers (70), indiCANCER RESEARCH VOL. 43 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1983 American Association for Cancer Research. Tamoxifen in Human Breast Cancer Table 1 Gìdistribution of control or tamoxifen-treated eating that accumulation of d cells with tamoxifen (as deter mined by analysis of DNA histograms) was not an artifact due to chromosome loss. for primary breast cancer with adjuvant antiestrogens may have to be continued indefinitely or at least long enough for host defense or normal cell attrition to eradicate residual tumor cells. Premature discontinuation of therapy would lead to tumor regrowth, (o) The combined use of antiestrogens with cytotoxic chemotherapy in an attempt to obtain an additive or synergistic effect will require careful evaluation. Tamoxifen might enhance the killing effect of drugs most active in Gìcells, whereas the cytotoxic effect of drugs acting specifically during DNA synthesis (S phase) might be diminished. This hypothesis is currently being investigated. DISCUSSION ACKNOWLEDGMENTS We have demonstrated that the antiestrogen, tamoxifen, inhib its proliferation of estrogen receptor-positive human breast can We would like to acknowledge the technical assistance of Philip Estrada, Sharon Olson, and Kathy Mosly. Earty G, (Stages 1-3) Control Tamoxifen 31 (38)" 49 (65) MCF-7 cells Late G, (Stages (4-6) 51 (62) 26 (35) p = 0.001 Numbers in parentheses, percentage of cells. cer cells. These results are similar to those reported by Lippman ef al. (9) and Sutherland ef al. (17) who found that, at concentra tions of tamoxifen observed in women treated for breast cancer (<1.0 UM), the antiestrogen slowed proliferation but was not lethal to MCF-7 cells in short-term culture. At suprapharmacological concentrations (10 MM), a cytocidal effect was observed (17), although the relevance of this effect to in vivo drug mech anism of action is speculative. The slight differences observed in the effects of tamoxifen in these studies may be related to differences in the type (charcoal stripped or not) and concentra tion of serum used. Our data suggest that tamoxifen inhibits cell proliferation by invoking a transition delay or block in early to mid-d phase of the cell cycle. This delay may require more than one complete passage of some cells through the cell cycle, since cell number more than doubled before plateau growth was observed, and since 72 to 96 hr (2 to 3 generation times) were required for maximal accumulation of cells in Gì.A small percentage of cells was refractory to tamoxifen and remained in the proliferative pool (5 to 10% in S phase). The mechanism of resistance of these cells to tamoxifen will require further study. We have shown recently that the tamoxifen effect can be reversed by the addition of 170-estradiol and that tamoxifen has no effect on the cell cycle kinetics of the receptor-negative MDA-MB-231 cells, suggesting that the antiestrogen effect is mediated through the estrogen receptor (13). Furthermore, these inhibitory effects are not restricted to tamoxifen. In our preliminary studies, the anties trogens nafoxidine and trioxifene have identical effects on breast cancer cell kinetics. These data are consistent with the hypothesis that antiestro gens, and perhaps other forms of endocrine therapy, induce regression of breast cancer in vivo by simply blocking progres sion of estrogen-dependent cells through the cell cycle rather than by a direct cytotoxic effect. With cell replication inhibited, tumors might then regress because of ongoing cell shedding or death or because of interaction with normal host defenses. This hypothesis would account for the very slow rate of tumor regres sion observed in many patients undergoing endocrine therapy. These results may have important clinical implications for the treatment of breast cancer, (a) If antiestrogens are simply putting endocrine-responsive breast cancer cells into a "resting" phase of the cell cycle (G0-Gi), then treatment of patients after surgery AUGUST 1983 REFERENCES 1. Alabaster, 0., and Bumag, B. Flow microfluorimetric analysis of sensitive and resistant leukemia L1210 following 1-/J-o-arabinofuranosylcytosine in vivo. Cancer Res., 36: 2744-2749,1976. 2. Dean P. N., and Jett, J. H. Mathematical analysis of DNA distributions derived from flow microfluorometry. J. Cell Biol., 60: 523-527,1974. 3. Edwards, D. P., Murthy, S. R., and McGuire, W. L. Effects of estrogen and antiestrogen on DNA polymerase in human breast cancer. Cancer Res., 40: 1722-1726,1980. 4. Hittelman, W. N., and Rao, P. N. Mapping Gt phase by the structural mor phology of the prematurely condensed chromosomes. J. Cell Physiol., 95: 333-342,1978. 5. Hittleman, W. N., Sognier, M. A., and Cole, A. Direct measurement of chro mosome damage and its repair by premature chromosome condensation. In: R. E. Meyn and H. R. Withers (eds.). Radiation Biology in Cancer Research, pp. 103-123. New York: Raven Press, 1980. 6. Horwitz, K. B., and McGuire, W. L. Antiestrogens: mechanism of action and effects in breast cancer. In: W. L. McGuire (ed.), Breast Cancer: Advances in Research and Treatment, pp. 155-204. New York: Plenum Publishing Corp., 1978. 7. Horwitz, K. B., and McGuire, W. L. Nuclear mechanisms of estrogen action: effects of estradiol and anti-estrogens on estrogen receptors and nuclear receptor processing. J. Biol. Chem., 253: 8185-8191,1978. 8. Jordan, V. C. Antiestrogenic and antitumor properties of tamoxifen in laboratory animals. Cancer Treat. Rep., 60: 1409-1419,1976. 9. Lippman, M., Bolán,G., and Huff, K. The effects of estrogens and antiestrogens on hormone-responsive human breast cancer in long-term tissue culture. Cancer Res., 36: 4595-4601,1976. 10. Lippman, M., Bolán. G., and Huff, K. Interactions of antiestrogens with human breast cancer in long term tissue culture. Cancer Treat. Rep., 60:1421-1429, 1976. 11. Livingston, R. B., Ambus, U., George, S. L., Freireich, E. J., and Hart, J. S. In vitro determination of thymidine-3H labeling index in human solid tumors. Cancer Res., 34:1376-1380,1974. 12. Osborne, C. K., and Boldt, D. H. Effects of antiestrogens on the cell cycle kinetics of cultured human breast cancer. J. Cell Biochem., (Suppl. 6V 1033A, 1982. 13. Osbome, C. K., Boldt, D. H.. Trent, J. M., and Clark, G. M. Synchronization of human breast cancer cells with antiestrogen block and estrogen rescue. In: Proceedings of the 64th Annual Meeting of the Endocrine Society, 56A, p. 93, 1982. 14. Osborne, C. K., Hamilton, B., Titus, G., and Livingston, R. B. Epidermal growth factor stimulation of human breast cancer cells in culture. Cancer Res., 40: 2361-2366,1980. 15. Sutherland, R. L., Murphy, L. C., Foo. M. S., Green, M. D., Whybourne, A. M., and Krozowski, Z. S. High-affinity anti-oestrogen binding site distinct from the oestrogen receptor. Nature (Lond.), 288: 273-275, 1980. 16. Sutherland, R. L.. and Taylor, I. W. Effect of tamoxifen on the cell cycle kinetics of cultured human mammary carcinoma cells. In: Reviews on Endocrine-related Cancer, Suppl. 8, pp. 8-15. ICI Pharmaceuticals Division, 1981. 17. Sutherland, R. L., Taylor, I. W.. Reddel, R. R., and Hall, R. E. Effects of tamoxifen on the cell cycle kinetics of human mammary carcinoma cells in culture. J. Cell. Biochem., Suppl. 6. p. 1034A, 1982. 18. Trent, J. Recent advances in cancer cytogenetics. Ariz. Med., 38: 836-838, 1981. 3585 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1983 American Association for Cancer Research. Effects of Tamoxifen on Human Breast Cancer Cell Cycle Kinetics: Accumulation of Cells in Early G 1 Phase C. Kent Osborne, David H. Boldt, Gary M. Clark, et al. Cancer Res 1983;43:3583-3585. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/43/8/3583 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1983 American Association for Cancer Research.