Involvement of PKA and Sp1 in the induction of p27(Kip1) by tamoxifen.
We have previously shown that tamoxifen (Tam) inhibits proliferation of estrogen receptor-negative human non-small cell lung cancer cells and this inhibition is associated with induction of p27(Kip1). In this study, we investigated the mechanism by which Tam increases p27(Kip1) expression. Because intracellular p27(Kip1) protein level is mainly controlled via posttranslational regulation, we first tested whether Tam might affect protein stability of p27(Kip1). Metabolic labeling and pulse chase assays showed that Tam did not affect the half-life of this protein. We next examined whether Tam enhanced p27(Kip1) expression through transcriptional activation. Our results demonstrated that Tam directly stimulated the p27(Kip1) promoter in lung cancer cells. Deletion and mutation analysis revealed that two Sp1 consensus sites located between -545 and -532bp from the transcription start site were crucial for the induction of p27(Kip1) by Tam. Conversely, mutation in a CTF site (-525/-520) nearby these two Sp1 sites had little effect. Electromobility shift assays showed that Sp1 transcription factor bound to these consensus sites and the DNA binding activity of Sp1 was enhanced by Tam. Our data also demonstrated that induction of p27(Kip1) by Tam was inhibited by protein kinase A inhibitor H89, but not by protein kinase C inhibitor calphostin C and mitogen-activated kinase kinase inhibitor PD98059. Taken together, our results suggest that Tam transcriptionally activates p27(Kip1) expression via the Sp1 consensus sites in the p27(Kip1) promoter and PKA is involved in this process.
Lee
Te-Hsiu
TH
Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan, ROC.
Chang
Hui-Chiu
HC
Chuang
Lea-Yea
LY
Hung
Wen-Chun
WC
D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology*; D018797:Cell Cycle Proteins / Q000096:biosynthesis* / Q000235:genetics; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D050760:Cyclin-Dependent Kinase Inhibitor p27; D015870:Gene Expression / Q000187:drug effects*; D006207:Half-Life; D006801:Humans; D011401:Promoter Regions, Genetic / Q000187:drug effects; D016329:Sp1 Transcription Factor / Q000502:physiology*; D013629:Tamoxifen / Q000494:pharmacology*; D015533:Transcriptional Activation / Q000187:drug effects*; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis* / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects
D018931:Antineoplastic Agents, Hormonal; D018797:Cell Cycle Proteins; D016329:Sp1 Transcription Factor; D025521:Tumor Suppressor Proteins; D013629:Tamoxifen; D050760:Cyclin-Dependent Kinase Inhibitor p27; D017868:Cyclic AMP-Dependent Protein Kinases