G1 arrest and expression of cyclin-dependent kinase inhibitors in tamoxifen-treated MCF-7 human breast cancer cells.
Treatment of exponentially growing MCF-7 human breast carcinoma cells with tamoxifen (TAM) inhibits cell growth in a dose-dependent manner. However, the molecular basis for the drug's activity and its relationship to the cell cycle have not yet been clearly established. In this study, we analyzed cell cycle-related proteins used for immunoblotting and flow cytometry in TAM-treated MCF-7 cells. In addition, the ratio of apoptosis in the cell was analyzed using labeling of DNA strand breaks (TdT assay). In flow-cytometric DNA distribution analysis, the S-phase fraction showed a marked decrease and a concomitant increase in G1- and G2-phase cells accompanying the inhibitory effect of TAM; these changes were time- and dose-dependent. Immunoblotting revealed that the levels of p53 and p21(WAF1/CIP1) in TAM-treated cells increased in a time- and dose-dependent manner, whereas those of p27(KIP1) and p16 slightly increased or remained unchanged. Furthermore, cyclin D3 and B showed sharp decreases, in contrast with p53 and p21(WAF1/CIP1) DNA-apoptosis dual analysis using flow cytometry revealed that the TAM-treated samples contained apoptotic cells, the majority of which were arrested in G1 or G2 and showed suppression of Bcl-2 protein. These results suggest that the tumorigenic effect of TAM on MCF-7 cells arises through antitumor effects that are due to the expression of cyclin-dependent kinase inhibitors, especially p21(WAF1/CIP1) and these are regulated by the decrease of wild-type p53. The proposed mechanism is similar to that underlying the cytotoxic effects of other agents and ionizing irradiation that cause DNA damage.
Ichikawa
Akira
A
Department of Laboratory Medicine, Tochigi Cancer Center, Utsunomiya, and Department of Pathology, Juntendo University School of Medicine, Tokyo, Japan. aichikaw@tcc.pref.tochigi.jp
Ando
Jiro
J
Suda
Koichi
K
D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology*; D017209:Apoptosis; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D002277:Carcinoma / Q000378:metabolism* / Q000473:pathology*; D050756:Cyclin-Dependent Kinase Inhibitor Proteins / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D016193:G1 Phase / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D013629:Tamoxifen / Q000494:pharmacology*; D013997:Time Factors; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D018931:Antineoplastic Agents, Hormonal; C493956:CDKN1A protein, human; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016159:Tumor Suppressor Protein p53; D013629:Tamoxifen