The gene expression of hepatic proteins responsible for DNA repair ...

00101.fm Page 582 Wednesday, July 2, 2003 3:29 PM

The gene expression of hepatic proteins responsible for DNA repair and cell proliferation in tamoxifen-induced hepatocarcinogenesis Toshihiko Kasahara,1 Chitose Kuwayama,2 Masamichi Hashiba,1 Tsuyoshi Harada,3 Chihaya Kakinuma,2 Makoto Miyauchi2 and Masakuni Degawa1, 4 1 Department of Molecular Toxicology and COE Program in the 21st Century, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, 2Department of Pathology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 and 3First Department of Biochemistry, Saitama Medical School, 38 Morohongo, Moroyama-machi, Iruma-gun, Saitama 350-0495

(Received March 4, 2003/Revised April 28, 2003/Accepted May 1, 2003)

Altered gene expression of the DNA repair- and cell proliferationassociated proteins/enzymes was examined during the process of tamoxifen-induced hepatocarcinogenesis in female Sprague-Dawley rats. When rats were treated by gavage with a single dose of tamoxifen (20 mg/kg body weight) or with the same dose given at 24-h intervals for 2, 12 or 52 weeks, no histopathological change was observed in the liver after 2 weeks. Pathologically altered cell foci and placental form of glutathione-S-transferase (GST-P)-positive foci were observed in the liver after 12 weeks of treatment. Treatment for 52 weeks resulted in the formation of liver hyperplastic nodules that strongly expressed GST-P. During the process of carcinogenesis, changes in hepatic gene expression of DNA repair proteins/enzymes (XPA and XPC, xeroderma pigmentosum complementation groups A and C, respectively; APE, apurinic/apyrimidinic endonuclease) and of cell proliferation-associated proteins (c-myc; PCNA, proliferating cell nuclear antigen; cyclin D1, cyclin B, and p34cdc2) were examined by RT-PCR. The gene expression of XPA and APE was increased by the tamoxifen treatment for 2 or 12 weeks, but no increase was observed after the 52-week treatment. In addition, no significant change in XPC gene expression occurred at any period examined. The gene expression of c-myc, PCNA, and cyclin D1 was increased in a timedependent fashion up to 12 weeks of treatment, and this increase was maintained up to 52 weeks of treatment. The gene expression of cyclin B and p34cdc2 was increased after the 1-day treatment, reverted to the control level at 2 and 12 weeks of treatment, and was remarkably increased after the 52-week treatment. In the present study, we demonstrate the altered gene expression of various proteins/enzymes involved in DNA repair, cell growth and the cell cycle during the process of tamoxifen-induced hepatocarcinogenesis. We discuss the relationship between the altered gene expression and hepatocarcinogenesis. (Cancer Sci 2003; 94: 582–588)

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otent hepatocarcinogenicity of the antiestrogen tamoxifen in rats has been reported,1–4) although tamoxifen is widely used not only as an anticancer drug,5, 6) but also as a chemopreventive agent7) for breast cancer. This hepatocarcinogenesis is thought to occur through the chemical modification of DNA by tamoxifen metabolites.8–13) α-(N 2-Deoxyguanosinyl)tamoxifen is detected as a major DNA adduct of tamoxifen in the rat liver, and the adduct has been shown to be formed through metabolic activations, α-hydroxylation and O-sulfation, of tamoxifen.14–21) Although N-desmethyltamoxifen, tamoxifen N-oxide, and 4-hydroxytamoxifen are major metabolites of tamoxifen, DNA adducts of these metabolites are hardly detected in the liver,19, 22) suggesting that these metabolites might be considered primarily as detoxification forms. Recently, we23) have investigated changes in the drug-metabolizing enzymes involved in forming tamoxifen metabolites 582–588

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during the process of tamoxifen-induced hepatocarcinogenesis and demonstrated that the level of CYP3A1, which is considered to be responsible for the activation (α-hydroxylation) of tamoxifen, increased from the early stage of formation of the liver hyperplastic nodules to the middle stage, and then decreased to the control level in the later stage. Furthermore, in the induced hyperplastic nodules, the sulfotransferase (HSTa) responsible for metabolic activation (O-sulfation of α-hydroxytamoxifen) of tamoxifen was decreased, while the flavin-containing monooxygenase 1 responsible for N-oxidation of tamoxifen, which is considered as a detoxification pathway, was markedly increased.23) Thus, overall changes in the gene expression of tamoxifen-metabolizing enzymes during tamoxifen treatment appear to be consistent with the formation and growth of hyperplastic nodules, because the increase in detoxification enzymes in the later stage would be expected to confer tamoxifen-resistance upon the induced nodules. It is generally considered that nucleotide excision repair (NER) serves to remove damage due to DNA adduct formation,24–26) and it is known that this repair system consists of two repair pathways, i.e., transcription-coupled repair (TCR) and global genome repair (GGR). XPA and XPC proteins, which are xeroderma pigmentosum complementation groups A and C, respectively, play an important role in the DNA repair systems; XPA for both the GGR and TCR pathways, and XPC for the GGR pathway.27) APE, the apurinic/apyrimidinic (AP) repair enzyme, and OGG1, the excision repair enzyme of 8-hydroxy-2′-deoxyguanosine (8-HOdG), are also involved in the repair of the corresponding DNA lesions.28, 29) Furthermore, OGG1 is induced by chemically generated DNA adducts.30) However, overall changes in expression of the DNA repair proteins/enzymes during the process of hepatocarcinogenesis have never been reported. Recently, Shibutani et al.31) reported that individual variations in repair capacity may be correlated with the development of tamoxifen-induced endometrial cancer, suggesting that DNA repair proteins/enzymes might play an important role in tamoxifen-induced hepatocarcinogenesis in female rats. Furthermore, since it is beyond doubt that cell proliferation-related proteins, c-myc,32–34) cyclin D1,35–38) PCNA,39, 40) p34cdc2 and cyclin B,41) are involved in chemically induced cell proliferation (cancerous cell formation), it is important to clarify changes in their expression levels during the process of carcinogenesis in order to understand the mechanism of chemical carcinogenesis. To date, however, overall changes in the expression of DNA repair- and cell growth/cycle-associated proteins/enzymes during the process of chemical carcinogenesis have not been 4 To whom correspondence should be addressed. E-mail: [email protected]

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reported. In the present study, therefore, we investigated changes in gene expression of the DNA repair proteins/enzymes (XPA, XPC, APE and OGG1) and of the cell growth/cycle-associated proteins (c-myc, cyclin D1, PCNA, p34cdc2 and cyclin B) during the process of tamoxifen-induced hepatocarcinogenesis in female rats. We discuss the relationship between the altered expression of these genes and hepatocarcinogenesis. Materials and Methods Treatment of animals. Female Sprague-Dawley rats were purchased from Charles River (Atsugi) and used at 6 weeks of age. They were kept individually in aluminum cages in an air-conditioned room and given CRF-1 diet (Oriental Yeast Co., Ltd., Tokyo) and water ad libitum. Tamoxifen citrate (Sigma Chemical Co., St. Louis, MO) was suspended in 0.5% carboxymethyl cellulose and used as tamoxifen in the present experiments. Treatments of rats with tamoxifen were performed as described previously.23) In brief, female rats were treated with a single daily dose (20 mg/kg body weight) by gavage for the indicated periods. The numbers of animals used were 14 for the 52-week treatment and 5 for other treatments. After the 52-week treatment, hyperplastic nodules were observed macroscopically in 12 of the 14 rats, and we selected 5 livers from these 12 animals for further experiments. In addition, the liver bearing hyperplastic nodules was divided into nodular and nodule-surrounding parts, and these were used as separate samples. The control group of 5 rats was treated with the vehicle (0.5% carboxymethyl cellulose solution) alone. Twenty-four hours after final treatment with tamoxifen or vehicle alone, the rats were sacrificed by exsanguination under anesthesia. Their livers were quickly removed and stored in liquid nitrogen until used. RT-PCR. Total hepatic RNA was prepared as described previously.23) Complementary DNA (cDNA) synthesis from total liver RNA (2 µg) was performed in a reaction mixture (40 µl) containing a NotI-(dT)18 primer (Amersham Pharmacia Biotech, Buckinghamshire, UK) and an “Omniscript” RT Kit (Qiagen GmbH, Hilden, Germany). Amplification of each cDNA (1 µl of the RT reaction mixture) was performed in a reaction mixture (50 µl) containing Gene Taq universal buffer (5 µl), dNTPs (each 0.2 mM), forward and reverse primers (each 20 pmol), and Gene Taq polymerase (1.25 units; Nippon Gene, Tokyo). Amplifications of all cDNAs examined were carried out with a PCR thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). The PCR program used was as follows: pretreatment at 94°C for 2 min; denaturation at 94°C for 20 s; annealing at 57°C for 45 s; and extension at 72°C for 45 s. The primer sets used and the predicted sizes of PCR products are summarized in Table 1. The primer sets for GST-P (a placental form of glutathione-Stransferase), XPA and XPC were designed by use of GenetyxMac software (Software Development Co., Ltd., Tokyo). Those

Table 1.

for XPA and XPC were prepared based on the mouse sequences. The sequences of rat XPA and XPC cDNAs obtained showed 95% and 94% homology, respectively, with the mouse ones (data not shown). A commercial primer set was used for G3PDH (Clontech Laboratories, Palo Alto, CA). The amount of product generated by each PCR increased linearly in a PCR cycle-dependent manner over the following ranges: 21–25 cycles for G3PDH; 25–29 cycles for GST-P, XPC and APE; 28–32 cycles for OGG1, PCNA; 30–34 cycles for XPA, c-myc and cyclin D1; and 32–36 cycles for cyclin B and p34cdc2. In addition, to assess possible contamination of the RNA preparation by genomic DNA, we performed direct PCR (without the RT reaction) of each RNA preparation. The PCR gave no product, demonstrating the absence of contamination with genomic DNA in the RNA preparations used. The expression level of each gene was measured as described previously.23) Briefly, a portion of each PCR product was applied to a capillary electrophoresis system (Hewlett-Packard Co., Wilmington, DE), and the separated PCR product was quantified spectrophotometrically in terms of the absorbance at 254 nm and normalized to that of G3PDH, the internal standard. Pathological analysis and immunohistochemistry. Paraffin sections (3-µm thickness) of the liver obtained from rats in each experimental group were made and placed on poly-L-lysine-coated slides. The sections were immersed in 3 changes of xylene and hydrated by using a graded series of alcohols. For histological study, sections were stained with hematoxylin and eosin (H&E). For immunohistochemical analysis of GST-P-positive foci, antigen retrieval was performed routinely by immersing the sections in distilled water in an autoclave for 10 min at 121°C. Endogenous peroxidase was quenched by treatment with 3% hydrogen peroxide in phosphate-buffered saline solution (PBS) for 10 min. Sections were blocked with 1% normal goat serum diluted with PBS at room temperature for 30 min and then incubated with polyclonal rabbit anti-rat GST-P (MBL, Nagoya) overnight at 4°C. After the sections had been washed with PBS, the following procedure was performed by using an “IMMU-MARK” universal immunostaining 500 Slide Kit (ICN ImmunoBiologicals, Cleveland, OH). The sections were incubated with biotin-labeled polyclonal goat anti-rabbit IgG at room temperature for 60 min. After a wash with PBS, the slides were incubated with horseradish peroxidase-labeled streptavidin at room temperature for 60 min and washed again. Peroxidase activity was then detected with 3,3′-diaminobenzidine as the substrate.49) Numbers of GST-P foci were counted on at least 1 cm2 of liver section from rats at each time point. Areas of sections were calculated by using an image analyzer (LUZEX III; Nireco, Tokyo), and the GST-P-positive foci generated were expressed as the number or percentage of area per cm2. In the liver of rats treated with tamoxifen for 52 weeks, GST-P-positive foci were not counted because so many had co-

Primer sequences and expected size of PCR products of target genes

Taeget gene GST-P XPA XPC APE OGG1 c-myc PCNA cyclin D1 cyclin B P34cdc2

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Primer sequences Forward 5′-TGGGTCGCTCTTTAGGGCTT-3′ 5′-AGCTGCAGAGATGCTGATGA-3′ 5′-TGTGCTGGACATGGGAGA-3′ 5′-CTGGACTTACATGATGAATGCCCG-3′ 5′-ATCTGTTCCTCCAACACCAAC-3′ 5′-AGTGCATTGATCCCTAGTGGTCTTTCCCTA-3′ 5′-GCCCTCAAAGACCTCATCAA-3′ 5′-TGGAGCCCCTGAAGAAGAG-3′ 5′-ACCTACTGGGTCGTGAAGTCACTGGAAA-3′ 5′-GAGAAAATCGGAGAAGGGACTTAT-3′

Reverse

Product size (bp)

Ref. No.

265 208 500 231 504 548 472 424 300 285

42 43 44 45 30 46 47 47 48 48

5′-AGCAGCAGGTCCAGCAAGTT-3′ 5′-AGCAGCAGGTCCAGCAAGTT-3′ 5′-GCAGAGCCCGAAGAATCA-3′ 5′-GAAGAGATAACGCACTGGTCTCCT-3′ 5′-GCCAGCATAAGGTCCCCACAG-3′ 5′-CAGCTCGGTTCCTCCTCTGACGTTCCAAGACGTT-3′ 5′-GCTCCCCACTCGCAGAAAAC-3′ 5′-AAGTGCGTTGTGCGGTAGC-3′ 5′-TCAGAATCTTCATCTCCATCTGTCTGAT-3′ 5′-AGAATCCATGAACTGGCCAGGAGGGA-3′

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Table 2. Histopathological and immunohistochemical analyses of the liver from rats treated with tamoxifen Treatment

No. of rats with altered cell foci

No. of rats with adenoma

No. of rats with carcinoma

GST-P-positive foci (No./cm2) 1)

Area of GST-P foci (%) 1)

0/5 0/5

0/5 0/5

0/5 0/5

ND ND

ND ND

0/5 0/5

0/5 0/5

0/5 0/5

ND ND

ND ND

0/5 0/5

0/5 0/5

0/5 0/5

0.7±0.7 10.9±9.3∗