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[Cancer Research 59, 2091-2095, May 1, 1999]
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[Cancer Research 59, 2091-2095, May 1, 1999]
© 1999 American Association for Cancer Research

Carcinogenesis

Mutagenic Potential of {alpha}-(N2-Deoxyguanosinyl)tamoxifen Lesions, the Major DNA Adducts Detected in Endometrial Tissues of Patients Treated with Tamoxifen1

Isamu Terashima, Naomi Suzuki and Shinya Shibutani2

Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Breast cancer patients treated with the antiestrogen tamoxifen (TAM) show an increased risk of developing endometrial cancer. We have recently detected TAM-DNA adducts in endometrium obtained from patients treated with TAM and identified them as trans- and cis-forms of {alpha}-(N2-deoxyguanosinyl)tamoxifen (dG-N2-TAM). To explore the mutagenic properties of these TAM-DNA adducts, we prepared site-specifically modified oligodeoxynucleotides containing a single isomer of dG-N2-TAM by reacting a 15-mer oligodeoxynucleotide containing a single dG (5'-TCCTCCTCGCCTCTC) with tamoxifen {alpha}-sulfate. These modified oligodeoxynucleotides were inserted into a single-stranded shuttle vector to investigate mutagenic specificities of the adducts in simian kidney (COS-7) cells. An epimer of dG-N2-trans-TAM showed targeted mutations ranging from 0.7 to 1.5%. The other dG-N2-trans-TAM adduct showed 9.6% G->T transversions, accompanied by 2.8% G->A transitions. Both dG-N2-cis-TAM adducts showed similar mutation spectra, where G->T transversions (11–12%) predominated, along with a small number of G->A transitions and G->C transversions. Thus, dG-N2-TAMs are mutagenic lesions in mammalian cells. The tamoxifen-DNA adducts detected in patient endometrium may cause mutations and initiate endometrial cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TAM3 is widely used for the chemotherapy of breast cancer. This drug is also currently undergoing clinical evaluation as a chemopreventive agent for healthy women at a high risk for the development of breast cancer (1, 2, 3) . The Breast Cancer Prevention Trial, initiated by the National Surgical Adjuvant Breast and Bowel Project, recently showed that TAM reduces the risk of both invasive and noninvasive breast cancer by 45–50% (4) , whereas the other TAM chemopreventive trial, initiated by the Royal Marsden Hospital, showed no significant reduction of breast cancer (5) . Approximately 50,000 women in the United States and Europe have been enrolled in the chemopreventive clinical studies (6, 7, 8) . However, treatment with TAM increases the incidence of endometrial cancer and second primary breast cancer in breast cancer patients (9, 10, 11) . The increased incidence of endometrial cancer was also observed in healthy women 50 years of age or older enrolled in the National Surgical Adjuvant Breast and Bowel Project TAM chemopreventive trial (4) .

TAM causes carcinomas in liver and uterus of rats (12, 13, 14) and promotes mutations in the liver of {lambda}/lac I transgenic rats (15) and in rat p53 gene in hepatocarcinomas induced by TAM (16) . Treatment with TAM produces DNA adducts in the livers of rodents (17 , 18) . Reactive species of TAM are formed by metabolic oxidation (19, 20, 21) , including {alpha}-hydroxylation of TAM and its analogues (22, 23, 24, 25) . Recently, sulfation of {alpha}-OH-TAM, catalyzed by rat and human hydroxysteroid sulfotransferases (26 , 27) , was shown to lead to the formation of trans- and cis-isomers of dG-N2-TAM (structures in Fig. 1Citation ; Refs. 28 and 29 ). These isomers interconvert via a short-lived carbocation intermediate (30) . dG-N2-TAMs are likely to be the major DNA adducts formed in rats treated with TAM or {alpha}-OH-TAM (31) . DNA adducts corresponding to 4-hydroxytamoxifen or tamoxifen 1,2-epoxide were not detected in these studies (31) , supporting the proposal that the primary route of TAM metabolism is by sulfation of {alpha}-hydroxylation.



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  		Fig. 1. Structures of trans- and cis-form of dG-N2-tamoxifen DNA adducts.

 
We recently found that dG-N2-TAM adducts have miscoding potential (32) . The miscoding specificities and frequencies varied, depending on the mammalian DNA pol used (32) . We also found that trans- and cis-isomers of dG-N2-TAM adducts can be detected in endometrium obtained from patients treated with TAM (33) . To explore the mutagenic specificity of dG-N2-TAM DNA adducts, oligodeoxynucleotides containing a single isomer of dG-N2-TAM were prepared by reacting a 15-mer oligodeoxynucleotide containing a single dG (5'-TCCTCCTCGCCTCTC) with tamoxifen {alpha}-sulfate {(Z)-1-{4-[2-(dimethylamino)ethoxy]phenyl}-1,2-diphenyl-1-buten-3-yl hydrogen sulfate} and inserted into a ss shuttle vector. These modified vectors were transfected into simian kidney (COS-7) cells. The progeny plasmid was used to transform Escherichia coli DH10B for analysis of mutations. We found that all dG-N2-TAM adducts were mutagenic, generating primary G->T transversions in mammalian cells. This indicates that these TAM-DNA adducts may cause mutations and initiate endometrial cancer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
[{gamma}-32P]ATP (specific activity, >6000 Ci/mmol) was obtained from Amersham Corp. (Arlington Heights, IL). E. coli DH10B was purchased from Life Technologies, Inc. (Grand Island, NY). The simian kidney (COS-7) cell line was obtained from Cold Spring Harbor Laboratory. EcoRI restriction endonuclease (100 units/µl) and T4 DNA ligase (400 units/µl) were obtained from New England Biolabs (Beverly, MA).

Preparation of Oligodeoxynucleotides.
An unmodified 15-mer oligodeoxynucleotide (5'-TCCTCCTCGCCTCTC) containing a single dG was prepared by solid-state synthesis, using an automated DNA synthesizer. As described previously (32) , 15-mer oligodeoxynucleotides containing a single dG-N2-TAM were prepared by reacting 100 µg of the 15-mer with 1.0 mg of tamoxifen {alpha}-sulfate for 2 h at 37°C in 500 µl of 50 mM sodium phosphate buffer (pH 7.0). After the reaction, the samples were extracted twice with 300 µl of butanol to remove TAM compounds. The aqueous fraction was evaporated to dryness, solved with 100 µl of distilled water, and subjected to HPLC. The unmodified and four isomers of dG-N2-TAM-modified oligomers were isolated on a reverse-phase µBondapak C18 (0.39 x 30 cm, Waters), using a linear gradient of 0.05 M triethylammonium acetate (pH 7.0) containing 10–30% acetonitrile, with an elution time of 60 min and a flow rate of 1.0 ml/min. These oligomers were further purified by electrophoresis on 20% polyacrylamide gel in the presence of 7 M urea (35 x 42 x 0.04 cm). A 990 HPLC instrument (Waters), equipped with a photodiode array detector, was used for separation and purification of oligodeoxynucleotides. The oligomers recovered from PAGE were again subjected to HPLC to remove urea. Oligomers were labeled at the 5' terminus by treating with T4 polynucleotide kinase in the presence of [{gamma}-32P]ATP (34) and subjected to electrophoresis to establish homogeneity. The position of the oligomers was established by autoradiography, using Kodak Xomat XAR film.

Site-specific Mutagenesis in COS-7 Cells.
SV40-transformed simian kidney cell line COS-7 and a ss vector, pMS2, which confers neomycin (NeoR) and ampicillin (AmpR) resistance (35) , were used to establish mutagenic specificity. Construction of a circular ss DNA containing a single DNA adduct followed procedures established previously in this laboratory (35) . pMS2 ss DNA was purified on a Nucleogen 4000–7 DEAE column (0.6 x 12.5 cm), using a linear gradient of 0.02 M potassium phosphate and 5 M urea (pH 6.9; eluent A) containing 40–100% eluent A and 1.5 M KCl (eluent B), with an elution time of 90 min and a flow rate of 1.0 ml/min. The fraction containing pMS2 (tR = 33.0 min) was concentrated on Centricon 100 filters, washed three times with distilled water, and subjected to ethanol precipitation. pMS2 DNA was annealed to a 61-mer and then digested with EcoRV to create a 15-mer gap (Fig. 2)Citation . An unmodified or dG-N2-TAM-modified 15-mer was ligated to the gapped vector. The ligation mixture was incubated for 2 h with T4 DNA pol (1 unit/pmol of DNA) to digest the hybridized 61-mer and then treated with EcoRV and SalI to cleave residual ss pMS2. The reaction mixture was extracted twice with phenol:chloroform, 1:1 (v/v), and twice with chloroform. Following ethanol precipitation, the DNA was dissolved in distilled water. A portion of the ligation mixture and known amounts of ss pMS2 were subjected to electrophoresis on a 0.9% agarose gel to separate closed circular and linear ss DNA. DNA was transferred to a nylon membrane and hybridized to a 32P-labeled S13 probe complementary to DNA containing the 15-mer insert. The absolute amount of closed circular ss DNA was established by comparing the radioactivity in the sample with that in known amounts of ss DNA.



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  		Fig. 2. Construction of a ss vector containing a single dG-N2-TAM DNA adduct. The upper strand is a part of ss pMS2 sequence. X, dG-N2-TAM. The underlined L13 and R13 probes were used to detect the correct insertion. The underlined 13-mer (S13) of 61-mer scafford (bottom strand) was used to determine the concentration of ss DNA construct. The probes listed were used for oligodeoxynucleotide hybridization to determine mutation specificity of dG-N2-TAM.

 
COS-7 cells were transfected with ss DNA (100 fmol) over 18 h using lipofection (36) , after which the cells were grown for 2 days in DMEM-10% FCS. Progeny plasmids were recovered by the method described by Hirt (37) , treated with S1 nuclease to digest input ss DNA, and used to transform E. coli DH10B. Transformants were analyzed for mutations by oligodeoxynucleotide hybridization (38 , 39) . The oligodeoxynucleotide probes used to identify progeny phagemids are shown in Fig. 2Citation . Probes L13 and R13 were used to select phagemids containing the correct insert. Transformants that failed to anneal with L13 and R13 were omitted from the analysis. When L13-/R13-positive transformants failed to hybridize to the probes designed to detect events targeted to the lesion site, double-stranded DNA was prepared and subjected to dideoxynucleotide sequencing analysis (40) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Preparation of Vectors Containing TAM-DNA Adducts.
A 15-mer oligomer containing a single dG was reacted with tamoxifen {alpha}-sulfate. Oligomers containing an epimer of trans-forms of dG-N2-TAM (tR = 28.6 and 30.5 min) or an epimer of the cis-forms (tR = 33.1 and 36.8 min) were isolated from the corresponding unmodified 15-mer (tR = 20.7 min) by HPLC (Fig. 3)Citation . As shown previously (32) , these modified oligomers were digested enzymatically, and incorporation of the trans- or cis-isomers of dG-N2-TAM in the oligomers was confirmed by HPLC, compared with the authentic dG-N2-TAM (data not shown). Because dG-N2-TAM has not yet been crystallized, the absolute configuration of the two trans-forms (trans-1 and trans-2) and the two cis-forms (cis-1 and cis-2) have not been determined (28) . These modified oligomers were purified twice by HPLC and by gel electrophoresis. Their homogeneities were confirmed after labeling with 32P (Fig. 4)Citation . The migration of dG-N2-TAM-modified 15-mers was much slower than that of the unmodified oligomer. These oligomers were ligated into a gapped ss vector, as shown in Fig. 2Citation . The final concentration of ss DNA was quantified by Southern blot hybridization. The S13 probe was hybridized to the ligation site of the ss vector (Fig. 2)Citation . Using a ß-phosphorimager, the net product of the closed circular DNA of each constructs was estimated by comparison with the variable amount of pMS2 DNA standards. The concentration of the closed circular vector sample was 4.5 ng/µl for the unmodified oligomer, and the concentrations of the trans-1, trans-2, cis-1, and cis-2 forms of dG-N2-TAM-modified oligomers were 5.3, 4.6, 5.6, and 7.9 ng/µl, respectively.



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  		Fig. 3. HPLC separation of 15-mer oligodeoxynucleotides containing a single dG-N2-TAM. One hundred µg of a 15-mer oligodeoxynucleotide (5'-TCCTCCTCGCCTCTC) were reacted with 1.0 mg of tamoxifen {alpha}-sulfate, as described in "Materials and Methods." The dG-N2-TAM-modified 15-mers were isolated on a µBondapak C18, using a linear gradient of 0.05 M triethylammonium acetate (pH 7.0), containing 10–30% acetonitrile with an elution time of 60 min and a flow rate of 1.0 ml/min.

 


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  		Fig. 4. PAGE of 15-mer containing a single dG-N2-TAM. Oligodeoxynucleotides were labeled with 32P, as described in "Materials and Methods," then subjected to electrophoresis on 20% polyacrylamide (15 x 72 x 0.04 cm).

 
Mutational Specificity.
The vectors modified with dG-N2-TAM were used to transfect COS-7 cells; the number of transformants recovered were compared to the unmodified vector (Table 1)Citation . A trans-isomer (trans-1) did not reduce transformation efficiency while the other isomers reduced it to 70–79%. When trans-1 of dG-N2-TAM-modified vector was used, dCMP (96.6%), the correct base was preferentially incorporated opposite the lesion (Table 2)Citation . A small number of mutations representing G->A transitions (1.5%) and G->T (1.1%) and G->C (0.7%) transversions were detected. In contrast, using the other trans-form (trans-2), a greater number of G->T transversions (9.6%) were observed, along with G->C transversions (2.8%). The frequency of mutation observed with trans-2 was 3.7 times higher than that of trans-1. On the other hand, cis-1 and cis-2 showed similar mutation frequencies and specificities. Both cis adducts showed G->T transversions (11–12%) predominantly, along with G->A transitions (1.7%). A small number of G->C transversions were detected only when cis-1 was used.


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  		Table 1 Transformation of COS-7 cells with ss DNA constructs

 

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  		Table 2 Mutational specificity of each stereoisomer of dG-N2-TAM DNA adducts in COS-7 cellsa

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A ss vector was used to establish mutagenic specificity for four stereoisomers of dG-N2-TAM replicated in COS-7 cells. One of the trans-forms (trans-2) and the two cis-forms (cis-1 and cis-2) showed a similar frequency (12–14%) of targeted mutations. G->T transversions (10–12%) were predominantly observed, along with small number of G->A transitions and/or G->C transversions. In contrast, the trans-1 promoted preferential incorporation of dCMP, the correct base, and showed lower mutational frequency (3.1%). Similar results were observed during in vitro DNA synthesis in reactions catalyzed by mammalian DNA pol {alpha}, ß, or {delta} (32) . When the trans-1 modified template was used, primers extended past the lesion much frequently than that of the other isomers and incorporated dCMP opposite the lesion (32) . This might be a reason why trans-1 isomer has low mutagenicity in COS-7 cells.

pol {alpha}, one of mammalian replicative enzymes, primarily misincorporated dAMP opposite all dG-N2-TAM adducts and additionally incorporated a small amount of dGMP. The predominant misincorporation of dAMP observed in vitro (32) was consistent with that observed in COS-7 cells. However, the frequency of G->T mutation (10–12%) observed opposite the trans-1, cis-1, or cis-2 lesion was much higher than that of dAMP misincorporation in vitro (1.5–2.3%). A small number of G->A mutations were detected in vivo, whereas no misincorporation of dTMP was observed with pol {alpha}. Using pol {delta}, another replicative enzyme, we detected a small amount of dTMP misincorporation only when trans-1 adduct was used (32) . These observations suggest that the mutagenic potential of dG-N2-TAM adducts may be modified by the presence of accessory proteins operating during translesional synthesis in mammalian cells (41) .

A limited number of TAM mutagenesis studies have been reported. When {lambda}/lacI transgenic rats were treated with TAM, significant amounts of G->T transversions were observed (15) . The mutation spectra were quite similar to our results. Because dG-N2-TAM is a major TAM DNA adduct in the liver of rats treated with TAM (31) , the mutations that occurred at G:C pairs in the liver of rats treated with TAM (15) are most likely due to dG-N2-TAM adducts.

On the other hand, A->G transitions, followed by C->T transitions, have been detected in the p53 gene in rat hepatocarcinomas induced by TAM (16) . The mutation spectra were quite different from that observed in the livers of {lambda}/lacI transgenic rats (15) and in our study. dA-N6-TAM can be produced by reacting DNA directly with an activated form of TAM, although the amount of dA-N6-TAM formed is much lower than that of dG-N2-TAM (42) . However, neither dA-N6-TAM nor dC-modified adducts have been detected in cells or in tissues of animals. Thus, A->G and C->T transitions are unlikely to be produced by TAM.

The same in vivo experimental system using COS-7 cells has been used for exploring mutagenic properties of 8-oxo-7,8-dehydroxy-2'-deoxyguanosine lesion, a typical form of oxidative DNA damage (35) , and DNA adducts derived from benzo[a]pyrene diol epoxide (43) , 2-AAF (44) , and model estrogen, 3-MeE (45) . Among these DNA adducts, dG-C8-AAF, dG-C8-AF, and dG-N2-3-MeE were located in the same sequence context as studied for the dG-N2-TAM adducts. Therefore, the mutagenic potential of dG-N2-TAM adducts can be compared with that of dG-C8-AAF, dG-C8-AF, or dG-N2-3-MeE adduct. Both dG-C8-AAF and dG-C8-AF adducts promoted preferential G->T mutations, along with a lesser amount of G->A mutations (44) . The mutational spectra are quite similar to those observed with dG-N2-TAM adducts. However, the mutational frequencies (12–14%) of dG-N2-TAM adducts except for the trans-1 are slightly higher than that of dG-C8-AAF (11%) and 4–5 times higher than that of dG-C8-AF (3.0%). dG-N2-3-MeE and dG-N2-TAM, N2-modified adducts, result in mostly G->T mutations (45) . The mutagenic frequencies of dG-N2-TAM adducts were ~2 times higher than that of dG-N2-3-MeE. Thus, the TAM-DNA adducts have much higher mutagenic potential than AAF- and estrogen-derived DNA adducts.

We have recently detected trans- and cis-isomers of dG-N2-TAM in the endometrial tissues obtained from patients treated with TAM (33) . The level of trans- and cis-form adducts tended to increase depending on duration of TAM therapy, ranging from 0.2 to 11.6 and 2.8 to 6.4 adducts per 108 nucleotides, respectively.4 In one case, a high level of TAM adducts was observed even after short-term TAM treatment. Because we have shown here that dG-N2-TAM adducts cause mutations and, therefore, may participate in the initiation of endometrial cancer, treatment of TAM may pose a potential risk to patients and healthy women participating in TAM chemopreventive trials.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by National Institute of Environmental Health Sciences Grant ES09418 (to S. S.). Back

2 To whom requests for reprints should be addressed, at Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-8651. Phone: (516) 444-8018; Fax: (516) 444-3218; E-mail: shinya{at}pharm.som.sunysb.edu Back

3 The abbreviations used are: TAM, tamoxifen; {alpha}-OH-TAM, {alpha}-hydroxytamoxifen; dG-N2-TAM, {alpha}-(N2-deoxyguanosinyl)tamoxifen; pol, polymerase; ss, single-stranded; HPLC, high-performance liquid chromatography; AAF, acetylaminofluorene; 3-MeE, 3-methoxyestra-1,3,5(10)-trien; dG-C8, N-(deoxyguanosin-8-yl); AF, aminofluorene; dG-N2-3-MeE, N2-[3-methoxyestra-1,3,5(10)-trien-6({alpha},ß)-yl]-2'-deoxyguanosine. Back

4 S. Shibutani, N. Suzuki, I. Terashima, S. Sugarman, A. P. Grollman, and M. Pearl, unpublished data. The preliminary data were presented in December 1998 in San Antonio, TX, at the 21st Annual San Antonio Breast Cancer Symposium. Back

Received 11/16/98. Accepted 3/ 4/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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