[CANCER RESEARCH54, 3357—3360,July 1, 19941

Advances in Brief

Tamoxifen Induces Hepatic Aneuploidy and Mitotic Spindle Disruption after aSingle in Vivo Administration to Female Sprague-Dawley Rats'

Linda M. Sargent, Yvonne P. Dragan, Nicole Bahnub, John E. Wiley, Carol A. Sattler, Pauline Schroeder,Gerald L SaWer, V. Craig Jordan, and Henry C. Pitot@McArdle Laboratory for Cancer Research, The Medical Schoo4 University of Wisconsin@Madison@ Wisconsin 53706 [L. M. S., Y. P. D., N. B., C. A. S., P. S., G. L. S., H. C. P.J;Medical Genetics@ East Carolina University Medical Schoo4 Greenville, North Carolina 27858 [J. E. W.J; and Lurie Cancer Center, Northwestern University, Chicago, Illinois60611 [V. C. J.J

Abstract

Tamoxifen has found extensive use in the treatment of all stages ofhuman breast cancer. The efficacy of tamoxifen treatment for the prevention of second primary tumors and Its chemosuppressive action In

animal models have led to Initiation ofclinical trials to test its emeacy forprevention of this disease In women. Recently, tamoxifen has been shown

to induce hepatocellular carcinomas in rats. For determination of themechanism of Induction of these tumors and assessment of the possibilityof risk of human cancer development from tamoxifen treatment, femaleSprague-Dawley rats (five rats per treatment) were administered tamoxIfen at doses ranging from 03 to 35 mgi'kg.One day after treatment, therats were sacrificed, sad the hepatocytes were isolated and cultured for50 h. Colcemid was added 3 h prior to harvest, and the hepatocytes werethen prepared for karyotypic evaluation. One hundred metaphase spreadswere examined per animaL Tamoxifen treatment resulted in the inductionof aneuploldy In approximately 70% of the examined hepatocytes at thedoses used. In addition, premature condensation (2—10%)and endoreduplication (5-10%) were observed in hepatocytes of rats treated withtamoxifen. Furthermore, exchanges between chromosomes as well aschromosomebreakage were observed.Examinationof the cultured hepatocytes from rats treated with tamoxifen by electron microscopy demonstrated both unipolar spindles and incompletely elongated splndles Exposure of rats to a single U?V1VOdOSeOf tamoxifen produced multiplechanges In rat hepatocytesIncludingclastogenicdamage at dosescomparable to that administered to humans. The occurrence of aneuploldyinduction, premature condensation, chromosome breakage, and Impropermitotic spindle formation IndIcates that risk versus benefit of tamoxifentreatment should be carefully evaluated.

Introduction

The antiestrogen, tamoxifen, has been used extensively to preventthe recurrence of human breast neoplasms, as well as the appearanceof further primary lesions (1). The efficacy of tamoxifen treatment forall stages of breast cancer (1, 2) and the low incidence of acute sideeffects associated with its use (3) have led to the institution oflarge-scale clinical trials using tamoxifen as a chemopreventive agentin individuals with an increased risk of breast cancer (4, 5). This useof tamoxifen for nonmalignant indications has prompted a reconsideration of the toxicology of tamoxifen. While studies on the toxicityof tamoxifen have not shown significant mutagenicity in microbialsystems (6), several experimental results suggest that tamoxifen maypossess a carcinogenic potential. Recent results from chronic bioas

Received 3/9/94; accepted 5/19/94.Thecostsof publicationof thisarticleweredefrayedinpartbythepaymentof page

charges.Thisarticlemustthereforebe herebymarkedadvertisementInaccordancewith18U.S.C.Section1734solelyto Indicatethisfact.

1This work was supportedin part by National Cancer Institute Grants CA-07175,CA-22484,andCA-57245,andby SpecialInstitutionalGrantSIO-15fromtheAmericanCancerSociety.

2To whom requestsfor reprints should be addressed,at McArdle Laboratory forCancerResearch,UniversityofWisconsln,1400UnIversityAvenue,Madison,Wisconsin53706.

says in rats have demonstrated an increased incidence of malignant

liver neoplasms compared with that in untreated animals (7). Tamoxifen is an effective promoting agent in hepatocarcinogenesis in the rat(8). Mani and Kupfer (9) have demonstrated that tamoxifen can forma reactive intermediate capable of binding to protein in a cell-freesystem. In addition, 32P-postlabeled DNA adducts can be detectedafter repeated doses of tamoxifen (10—12).Further studies have demonstrated that tamoxifen can induce a G@block in MCF-7 cells (13).Francavila et a!. (14) have demonstrated that tamoxifen can inhibithepatic proliferation after a partial hepatectomy, suggesting that thisagent also induces a G@block in hepatocytes. Tamoxifen induces theformation of micronuclei in a metabolism-competent lymphoblastoidcell line (1 1, 12). Because of the striking carcinogenic effect oftamoxifen on rat liver in vivo, its structural similarity to DES,3 and itsability to induce micronuclei, the effects of tamoxifen on hepaticploidy and chromosomal integrity were investigated to determinewhether either of these types of genetic damage might be integral tothe carcinogenic action of tamoxifen.

Materials and Methods

In order to determine whether tamoxifen induced aneuploidy or chromosome damage in rat liver in vivo, we administered tamoxifen citrate (SigmaChemical Co., St. Louis, MO) to groups containing five female SpragueDawley rats (Harlan Sprague-Dawley, Madison, WI) in doses of 0.3, 3.0, and35.0 mg/kg by gavage. Alternatively, the solvent, trioctanoin (Pfaltz andBauer, Waterbury, CT), or the positive control for induction of aneuploidy(15), 35 mg TCPOBOP, synthesized and provided as a gift by Dr. JamesMiller, McArdle Laboratory, was administered. Twenty-four hours after dosing, the rat livers were perfused with a collagenase solution as describedpreviously (16). Hepatocytes were separated from littoral cells by a Percoll(Pharmacia, LKB Biotechnology, Piscataway, NJ) isodensity centrifugationand immediately plated in 75-cm collagen-coated flasks (16) at a density of2.6 X@ cells in 15 ml of serum-free “supplemented―Dulbecco's modifiedEagle's medium/Ham's F-12 medium (17). The medium was supplementedwith bovine serum albumin (0.2%), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (18 mM), and sodium pyruvate (5 mM). The medium was changedafter 3 h, and 10 ng epidermal growth factor (Collaborative BiomedicalProducts, Bedford, MA) was added per ml of medium to stimulate celldivision. After 47 h ofculture, 40 ng/ml Colcemid was added to each flask, andthe cells were incubated for an additional 3 h. Parallel cultures were treatedwith 25 g.LM5-bromo-2'-deoxyuridine (Sigma) for the last 6 h of culture topermit analysis of late replication banding (18). Cultures were randomlyselected, andthe cells were preparedfor examinationby electronmicroscopyby methods described previously (19) or harvested for karyotypic analysis. Thehepatocytes were removed from the dish with 0.01% collagenase and harvestedby hypotonictreatment(0.075MKC1)for 8.5 mm. Once removedfrom theflask, the hepatocytcs were fixed with mcthanol:acctic acid (3:1, v/v), andslides were prepared as described by Sargent et a!. (20). One hundred metaphases of good morphology were randomly selected from cultures of each rat.

3The abbreviationsused are: DES, diethylstilbestrol;TCPOBOP, 1,4-bis[2-(3,5-dlch1omnvridvlnxv@1benzene.

3357

on March 30, 2020. © 1994 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

Table 1 Effect of a single oral dose of tamoxifen on rat hepatic chromosomal number and integrityAverage percentage of hepatocytes in cultures from five rats at each dose ±SD demonstrating chromosome breakage and other aberrations after treatment in vivo with tamoxifen

in trioctanoin at 0.3—35mg/kg administered in vivo by gavage. Values for hepatocyte cultures from five rats receiving the solvent only or TCPOBOP are also given. TCPOBOP wasadministered at a dose of 35 mg/kg by gavage (23).

PrematureTreatmentAneuploidyBreakageCondensationExchangesEndoreduplicationSolvent

control3 ±3.05.0 ±2.0<1.0<1.0<1.035

mg TCPOBOP/kg65 ±6.5a10.0 ±5.0―<1.0<1.0<1.00.3

mg Tam/kg3 mg Tam/kg71

±8.0°70 ±5.0―8.0

±2.09.4 ±3.02.0

±1.04.0 ±2.5―8.0

±3.0―5.0 ±2.0―5.0

±1.0―6.0 ±2.0―35

mg Tam/kg85 ±7.0―23.0 ±4.0―10.0 ±5.0―10.0 ±2.0―10.0 ±2.0―a

Significant difference from thecontrol treatment (P < 0.05); Tam, tamoxifen.

TAMOXIFEN INDUCES HEPA11C ANEUPLOIDY IN VIVO

than 0.3 mg/kg may be necessary to demonstrate a dose-dependenteffect for induction of aneuploidy by tamoxifen. Tamoxifen treatmentinduced a dose-related increase in premature condensation and chromosome breakage (Table 1) that was significantly greater than thatobserved in solvent-treated rats (P < 0.05). Additionally, a dosedependent induction of endoreduplication was observed with tamoxifen treatment. Furthermore, a significant level (P < 0.05) of aneuploidy, chromosomal exchanges, and endoreduplication wereobserved for all doses of tamoxifen administered (Table 1).

Fig. 1 is a photomicrograph of a metaphase of a type typicallyobserved in rats treated with 35 mg tamoxifen citrate/kg. The spreadis aneuploid. The presence of diplochromosomes (paired homologouschromosomes) indicates that the spread has undergone endoreduplication. In addition, chromosomes without a homologue can be observed (Fig. 1, arrow). Also, two exchanges are shown in this metsphase spread (Fig. 1, double arrows). All of the observed exchangeswere between homologous chromosomes, indicating that the cxchanges occurred during S synthesis (21).

Since the mechanisms of induction of aneuploidy and endoreduplication can be quite varied (22), two factors potentially responsible forthese effects were examined. In order to determine whether thetamoxifen-induced aneuploidy and endoreduplication are associatedwith an inhibition of chromosome synthesis, we examined metaphasespreads by late replication banding (18). In these studies, all metaphase spreads investigated showed a normal banding pattern with alight-staining, late-replicating X chromosome (data not shown), mdi

S

1'•

In the animals examined, approximately 70% of the hepatocytes were tetraploid (chromosome number is 84), and 30% were diploid (chromosomenumber is 42). A spread was considered aneuploid if it deviated from theestablished diploid or tetraploid number of chromosomes. The metaphaseswere analyzed blindly for ploidy, breakage, exchanges, premature condensetion, and endoreduplication. The significance of differences between groupswas determined by nonparametric@ statistics; evidence of a dose-responserelationship was tested by regression analysis. The alpha level was adjusted totake into account the number of comparisons.

Results

Karyotypic analysis of metaphase spreads from Sprague-Dawleyfemale rats was performed to determine the effect of a single tamoxlien treatment on the number and integrity of hepatic chromosomes.Only a low level of breakage and aneuploidy similar to that reportedpreviously for control untreated rats was observed in the solventtreated rats (Table 1). TCPOBOP, which has previously been shownto induce aneuploidy in the rat liver in vivo (15), was used as apositive control to demonstrate that aneuploidy could be detected withthis model. The present study showed that 35 mg TCPOBOP/kg bodyweight resulted in substantial aneuploidy (Table 1). Tamoxifen alsoinduced a marked level of aneuploidy compared with the solventcontrol (P < 0.05). The level of aneuploidy was extremely highcompared with the control. Aneuploidy was significantly increased forall doses of tamoxifen tested (P < 0.05). Since the level of aneuploidynoted was independent of the dose of tamoxifen used, doses lower

Fig. 1. A hepatic metaphase spread prepared froma female Sprague-Dawley rat administered 35 mg/kgtamoxifen citrate and sacrificed 24 h later is shown.This metaphase spread demonstrates aneuploidy, cxchanges, and endoreduplication. The two exchangesare indicated by double arrows, while the three chromosomes missing a homologue are indicated by single arrows. Hepatocytes were isolated by perfusionand Percoll isodensity centrifugation before cultureand preparation for cytogenetic analysis. Further details on the methods are described in Table 1 and in“Materialsand Methods.―

F

&

a

3358

4:

on March 30, 2020. © 1994 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

TAMOXIFEN INDUCES HEPATIC ANEUPLOIDY IN VIVO

eating that the observed aneuploidy and endoreduplication were notdue to a block in S synthesis.

Examination of hepatic metaphase spreads from solvent- and tamoxifen-treated rats by electron microscopy shows some interestingdifferences in mitotic spindle integrity (Fig. 2). Bipolar spindles werenoted in control cultures (Fig. 2A), while in a significant number ofcultures from tamoxifen-treated rats, monopolar spindles (Fig. 2B)were observed. Of the 36 informative spindles examined, 9 weremonopolar. In addition, the organelles were not excluded from thespindle in a number of these spreads. In some tamoxifen-treatedhepatocytes (data not shown), a normal bipolar spindle appeared tohave formed, but during anaphase the spindle elongation was muchless than in untreated hepatocytes. Thus, while tamoxifen treatmentdoes not appear to affect S phase DNA synthesis, there is clearly an

Fig. 2. Electron micrograph ofa dividing hepatocyte prepared from a Sprague-Dawleyrat followingtrioctanoinintubation(A)or treatmentwith3 mg/kgtamoxifenCitrate(B).Themetaphasespreadin (A)hasa spindleapparatusof normallengthandcontainstwopoles. The metaphase cell in (B) contains a monopolar spindle. In addition, the organellesarc not excluded from the metaphase plate in the spread provided in (B). B, arrow, thesingle pair of centrioles in this spindle apparatus.

effect of the in vivo administration of this agent on the structure of themitotic apparatus that could lead to aneuploidy (22).

Discussion

The administration of tamoxifen induces premature condensation ofthe hepatic chromosomes, chromosome exchanges, and chromosomebreaks. Compounds structurally related to tamoxifen can block cellcycle progression (23). In addition, another structural analogue oftamoxifen, DES, induces mitotic arrest (24). Tamoxifen induces a G@block in MCF-7 cells (13) and possibly in hepatocytes in vivo (14).Since the blockage of cell cycle progression can result in prematurecondensation, this action of tamoxifen should be explored to uncoverthe mechanism by which tamoxifen causes premature condensation.The presence of tamoxifen-induced DNA adducts (10—12)may resultin premature condensation, exchanges between chromosomes, andchromosomal breakage. Since the exchanges were observed in thisstudy only between homologous chromosomes, the exchanges cccurred during S (21). Thus, tamoxifen-induced DNA damage resultsin changes in cell cycle progression and further karyotypic instability.

Tamoxifen administration in vivo results in aneuploidy, an alteration in the number of chromosomes, and endoreduplication, which isthe result of two or more synthesis cycles in the absence of anintervening mitosis. A block in DNA synthesis or the inhibition ordelay of spindle formation can result in endocycles and in aneuploidy.The presence of a light-staining, late-replicating X chromosome indicates that the endoreduplication and aneuploidy that occurred aftertamoxifen treatment were not due to a block in S synthesis (18). Thepresence of monopolar spindles and incompletely elongated spindlesfrom which the organelles were not excluded suggests that the aneuploidy and endoreduplication observed with tamoxifen treatment maybe due to the induction of spindle aberrations. A structural analogueof tamoxifen, DES, has also been shown to induce micronuclei (25),aneuploidy (26), and cellular transformation (27). In addition, DEStreated cells have been shown to undergo mitotic arrest (24) and tocontain monopolar spindles (28). Therefore, this class of agents appears to have multiple effects on the cell that result in the disruptionof the normally tightly regulated coordination of spindle formationand chromosomal integrity.

Multiple factors are involved in the maintenance and proper formation of the mitotic spindle apparatus. Although the mechanism bywhich tamoxifen induces aneuploidy and karyotypic instability isunknown, tamoxifen is known to alter cellular calcium levels (29) andacts as a calmodulin antagonist (30). Although the role of calmodulinin spindle assembly is not known, calmodulin is associated with thespindle pole body and is required for proper formation of the spindleapparatus (31). Calmodulin-defective mutants in yeast contain a singlespindle pole body, monopolar spindles, and shortened spindles thatappear not to have elongated (31). The phenotypes associated withthese calmodulin mutants include aneuploidy in some of the cells, aswell as a low percentage of cells with endoreduplication (32). Sincethese phenotypes are similar to the observed effects of tamoxifen, theaneuploidy and endoreduplication observed in the rat may be due toinhibition of the action of calmodulin. Furthermore, yeast mutants thathave improper spindle pole body formation also contain monopolarspindles (31, 32), as was observed in the present study in rat hepatocytes, further supporting the possibility that tamoxifen may alter boththe number and integrity of chromosomes through its inhibitory actionon calmodulin or one of the spindle pole body proteins.

The effectiveness of tamoxifen as a clastogenic agent indicatesthat tamoxifen induces changes in both the number and structuralintegrity of the chromosomes and may increase the progression ofcells toward frank neoplasia. Aneuploidy and endoreduplication

3359

“@ .p.@.. .@_n.••.p@@

on March 30, 2020. © 1994 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

TAMOXIFEN INDUCES HEPATIC ANEUPLOIDY IN VJVO

are often associated with malignant conversion (33). In addition,the hallmark of the stage of progression is one of an evolvingkaryotypic instability, including an increase in aneuploidy andchromosome breakage with gene deletions, rearrangements, andduplications (34). Since tamoxifen possesses clastogenic action asevidenced by the induction of micronuclei, aneuploidy, and chromosomal breakage, its carcinogenic action may include effectiveness during the stage of progression. This supposition is supportedby the induction of transformation of Syrian hamster embryo cellsby tamoxifen (35). Retrospective studies have found an increasedincidence of endometrial cancers in tamoxifen-treated women (36).The present study performed in rat hepatocytes indicates thattamoxifen alters the number of chromosomes, increases the mcidence of chromosomal aberrations, and may disrupt mitotic spindleintegrity. The carcinogenic action of tamoxifen for the rat liver,coupled with the occurrence of aggressive breast and uterinetumors, albeit at a low incidence in women treated with tamoxifen,suggests that a careful assessment of the risk as well as the benefitof chronic tamoxifen administration should be considered in thenonmalignant use of this drug.

References

1. Jordan, V. C. A current view of tamoxifen for the treatment and prevention of breastcancer. Br. J. Pharmacol., 110: 507—517,1993.

2. Jordan, V. C., and Murphy, C. S. Endocrine pharmacology of antiestrogens asantitumor agents. Endocrine Rev., 11: 578—610,1990.

3. Legha, S., and Carter, S. Antiestrogens in the treatment of breast cancer. CancerTreat. Rev., 3: 205—216,1976.

4. Powles, T., Tillyer, C., Jones, A., Ashley, S., TreLeavan, J., Davey, J., and McKinna,J. Prevention of breast cancer with tamoxifen—anupdate on the Royal MarsdenHospital pilot programme. Eur. J. Cancer, 26: 680-684, 1990.

5. Love, R. R. Tamoxifen prophylaxis in breast cancer. Oncology, 6: 33—43,1992.6. Tucker, M. J., Adam, H. K, and Patterson, J. S. Tamoxifen. In: D. R. Laurence,

A. E. M. Mclean, and M. Weatherall (eds.), Safety Testing of New Drugs, pp.125—161.New York: Academic Press, 1984.

7. Williams, 0. M., latropoulos, M. J., Djordjevic, M. V., and Kaltenberg, 0. P. Thetriphenylethylene drug tamoxifen is a strong liver carcinogen in the rat. Carcinogenesis (Land.), 14: 315—317,1993.

8. Yager, J. D., Roebuck, B. D., Paluszcyk, T. L, and Memoli, V. A. Effects of ethinylestradiol and tamoxifen on liver DNA turnover and new synthesis and appearance ofy-glutamyl transpeptidase-positive foci in female rats. Carcinogenesis (Land.), 7:2007—2014,1986.

9. Mani, C., and Kupfer, D. Cytochrome P450 mediated action and irreversible bindingof the antiestrogen tamoxifen to proteins in rat and human liver: possible involvementof the flavin-containing monooxygenases in tamoxifen activation. Cancer Rca., 51:6052—6058,1991.

10. Han, Y., and Liehr, J. 0. Induction of covalent DNA adducts in rodents by tamoxifen.Cancer Res., 52: 1360-1363, 1992.

11. White, I. N. H., de Matteis, F., Davies, A., Smith, L L, Crofton-Sleigh, C., Venitt,S., Hewer, A., Phillips, D. H. Genotoxic potential of tamoxifen and analogues infemale Fischer F344/N rats, DBA/2 and C57BL/6 mice and human MCL-5 cells.Carcinogenesis, 13: 2197—2203,1993.

12. Styles, i. A., Davies, A., Urn, C. K, de Matteis, F., Stanley, L A., White, I. N. H.,Yuan, Z.-X., Smith, L. L. Genotoxicity of tamoxifen, tamoxifen epoxide andtoremifene in human lymphoblastoid cells containing human cytochrome P450s.Carcinogenesis, 15: 5—9,1994.

13. Osborne, C., Boldt, D., and Estrada, P. Human breast cancer cell cycle synchronization by estrogens and antiestrogens in culture. Cancer Res., 44: 1433—1439,1984.

14. Francavilla, A., Polimeno, L., Dileo, A., Barone, M., Dye, P., Coetzee, M., Eagon, P.,Makowa, L, Ambrosino, 0., Mazzaferro, V., and Starzl, 1. The effect of estrogen and

tamoxifen on hepatocyte proliferation in vivo and in vitro. Hepatology, 9: 614—620,1989.

15. Rossi, A. M., Zaccaro, L, Rosselli, F., and Quatirone, C. Clastogenic effects inducedin mice and rats by 1,4-bis[2-(3,5-dichloropyridyloxy)Jbenzene,a phenobarbital-likeenzyme inducer and liver tumour promoter. Carcinogenesis (Lond.), 9: 1147—1151,1988.

16. Xu, Y-H., Sattler, G. L., and Pitot, H. C. A method for the comparative study ofreplicative DNA synthesis in GOT-positive and GOT-negative hepatocytes in primaryculture isolated from carcinogen-treated rats. In Vitro Cell. Dcv. Biol., 24: 995—1000,1988.

17. Li, Y., Sattler, 0., and Pitot, H. Oxaloacetate induces DNA synthesis and mitosis inprimary cultured rat hepatocytes in the absence of EGF. Biochem. Biophys. Res.Commun., 193: 1339—1346, 1993.

18. Huret, J., Delabar, J., Marlens, F., Aurias, A., Tanger, J., and Sinet, D. Downssyndrome with duplication of a region of chromosome 21 containing the CuZnsuperoxide dismutase gene without detectable karyotypic abnormalities. Hum. Genet.,75: 251—257,1987.

19. Sattler, C. A., Sawada, N., Sattler, G. L, and Pitot, H. C. Electron microscopic andtime lapse studies of mitosis in cultured rat hepatocytes. Hepatology, 8: 1540—1549,1988.

20. Sargent, L, Xu, Y-h., Sattler, 0. L, Meisner, L, and Pitot, H. C. Ploidy andkaryotype of hepatocytes isolated from enzyme-altered foci in two different protocolsof multistage hepatocarcinogenesis in the rat. Carcinogenesis (Lond.), 10: 387—391,1989.

21. DiPaolo, J., and Popescu, N. Banding pattern analysis of initial structural chromosome alterations induced by N-methyl-N-nitroso-N-nitmsoquinone in Syrian hamstercells. Mutat. Res., 44: 359-368, 1977.

22. Paweletz, N., Ghosh, S., and Vig, B. K. Mitosis and the induction of aneuploidy. In:

Mechanisms of Chromosome Distribution and Aneuploidy, pp. 217—229.Alan R.1155, Inc., 1989.

23. Baker, W., Maenpaa, J., Wurz, G., Koester, S., Seymour, R., Emshoff, V., Wiebe, V.,and DeGregorio, M. Toremifene enhances cell cycle block and growth inhibition byvinblastine in multidrug resistant human breast cancer cells. Oncology Res., 3:207—212,1993.

24. Hill, A., and Wolff, S. Sister chromatid exchanges and cell division delays induced byDES, estradiol, and estriol in human lymphocytes. Cancer Res., 43: 4114—4118,1983.

25. Schiffman, D., and Bon, U. Dislocation of chromatin elements in prophage inducedby diethylstilbesterol: a novel mechanism by which micronuclei can arise. Mutat.Res., 246: 113—122, 1991.

26. Tsutsui, T., Maizumi, H., McLachlan, J., and Barrett, J. Aneuploidy induction and celltransformation by DES: a possible chromosomal mechanism in carcinogenesis@CancerRca.,43:3814—3821,1983.

27. Barrett, J., Wong, A., and McLachlan, J. DES induces neoplastic transformation

without measurable gene mutation at two loci. Science (Washington DC), 212:1402—1404,1981.

28. Liang, J. C., and Brinkley, B. R. Chemical probes and possible targets for theinduction of aneuploidy. Basic Life Sd., 36: 491—505,1985.

29. Upton, A., and Morris, I. Calcium antagonism by the antiestrogen tamoxifen. CancerChemother.Pharmacol.,18: 17—20,1986.

30. Edwards, K., Laughton, C., Neidle, S. A molecular modeling study of the interactionsbetween the antiestrogen drug tamoxifen and several derivatives, and the calciumbinding protein calmodulin. J. Med. Chem., 35: 2753—2761,1992.

31. Geiser, J. R., Sundberg, H. A., Chang, B. H., Muller, E. G. D., and Davis, T. N. Theessential mitotic target of calmodulin is the 110-kilodalton component of the spindlepole body in Saccharomyces cerevisiae. Mol. Cell. Biol., 13: 7913—7924,1993.

32. Sun, G-H., Hirata, A., Ohya, Y., and Anraku, Y. Mutations in yeast calmodulin causedefects in spindle pole body functions and nuclear integrity. J. Cell BioL, 119:1625—1639, 1992.

33. Aldaz, C., Conti, C., Klein-Szanto, A., and Slaga, 1. Progressive dysplasia andaneuploidy are hallmarks of mouse skin papillomas: relevance to malignancy. Proc.Nail. Mad. Sd. USA, 84:2029-2032,1987.

34. Pitot, H. Progression: the terminal stage in carcinogenesis. Jpn. J. Cancer Res., 80:599—607, 1989.

35. Metzler, M., and Schiffman, D. Structural requirements of the in vitro transformationof Syrianhamsterembryocells by stilbeneestrogensand triphenylethylene-typeantiestrogens. Am. J. Chin.Oncol., 4 (Suppl. 2): 530—535,1991.

36. Seoud, M. A-F., Johnson, J., and Weed, J. C., Jr. Gynecologic tumors in tamoxifentreated women with breast cancer. Obstet. Gynecol., 82: 165—169,1993.

3360

on March 30, 2020. © 1994 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

1994;54:3357-3360. Cancer Res   Linda M. Sargent, Yvonne P. Dragan, Nicole Bahnub, et al.   Sprague-Dawley Rats

Administration to Femalein VivoDisruption after a Single Tamoxifen Induces Hepatic Aneuploidy and Mitotic Spindle

  Updated version

  http://cancerres.aacrjournals.org/content/54/13/3357

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/54/13/3357To request permission to re-use all or part of this article, use this link

on March 30, 2020. © 1994 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from