The gene expression of hepatic proteins responsible for DNA repair and cell proliferation in tamoxifen‐induced hepatocarcinogenesis

Abstract

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) otent hepatocarcinogenicity of the antiestrogen tamoxifen in rats has been reported, [1][2][3][4] although tamoxifen is widely used not only as an anticancer drug, 5,6) but also as a chemopreventive agent 7) 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][15][16][17][18][19][20][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, we 23) have investigated changes in the drug-metabolizing enzymes involved in forming tamoxifen metabolites during the process of tamoxifen-induced hepa...

Increased Expression of Cyclin-Dependent Kinase-Interacting Protein p21 during Tamoxifen-Induced Hepatocarcinogenesis in Female Rats

“…In addition, such p53-independent expression of p21 has been reported to occur in muscle and other terminally differentiating cells. 17) During the initiation-promotion process (~12 weeks) of tamoxifen-induced hepatocarcinogenesis, the expression pattern of p21, which promotes apoptosis and DNA repair by arresting the cell cycle in DNA-damaged cells, 18) was similar to those of tamoxifen-activating enzyme(s), especially the CYP3A subfamily enzymes and DNA repair enzyme, 9) suggesting that the increased gene expression of p21 was dependent on the development of tamoxifen-induced DNA damage in liver cells.…”

“…8,9) Briefly, female Sprague-Dawley rats were purchased from Charles River (Atsugi, Japan) and used at 6 weeks of age. Tamoxifen citrate (Sigma Chemical Co., St. Louis, MO, U.S.A.) was suspended in 0.5% carboxymethyl cellulose and used as tamoxifen.…”

“…9) Briefly, synthesis of complementary DNA (cDNA) from total liver RNA (2 µg) was performed in a reaction mixture (40 µl) containing a Not I-(dT)18 primer (Amersham Pharmacia Biotech, Buckinghamshire, U.K.) and an Omniscript RT Kit (Qiagen GmbH, Hilden, Germany). Preparation of Standard DNAs for Real-Time PCR ---Standard DNAs for the quantitative determination of target mRNAs using real-time PCR were amplified with the cDNAs synthesized from total RNAs of untreated rat liver with the Omniscript RT Kit.…”

“…9) Immunohistochemical analysis of p21 was performed according to the following protocol. After endogenous peroxidase in a liver section was quenched by treatment with 3% hydrogen peroxide in phosphate-buffered saline (PBS) solution for 10 min, the section was immersed in 5% normal goat serum (IBL Co., Ltd., Gunma, Japan) at room temperature for 30 min, rinsed with Tris-buffered saline solution containing 0.1% Tween20 (TBS-T), and then incubated with a mouse anti-human p21 WAF1/Clip1 monoclonal antibody (DakoCytomation Co., Ltd., Glostrup, Denmark) overnight at 4°C.…”

“…7) Recently, we have demonstrated the alteration of the gene expression of the tamoxifen-metabolizing enzymes, 8) DNA repair proteins/enzymes, and cell proliferation-associated proteins during the process of tamoxifen-induced hepatocarcinogenesis in female rats and discussed the correlation of those alterations with the development of hepatocarcinoma. 9) In tumor biology, cyclin-dependent kinase-interacting protein p21 (p21) has received attention because it suppresses the cell cycle progression by inhibiting the activity of cyclin-dependent kinases 10,11) and by interacting with a proliferation cell nuclear antigen (PCNA). 12) Considering these functions of p21, it appears to be closely associated with the prevention of carcinogenesis.…”