p21(WAF1/CIP1) is upregulated by the geranylgeranyltransferase I inhibitor GGTI-298 through a transforming growth factor beta- and Sp1-responsive element: involvement of the small GTPase rhoA.
We have recently reported that the geranylgeranyltransferase I inhibitor GGTI-298 arrests human tumor cells at the G1 phase of the cell cycle and increases the protein and RNA levels of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). Here, we show that GGTI-298 acts at the transcriptional level to induce p21(WAF1/CIP1) in a human pancreatic carcinoma cell line, Panc-1. This upregulation of p21(WAF1/CIP1) promoter was selective, since GGTI-298 inhibited serum responsive element- and E2F-mediated transcription. A functional analysis of the p21(WAF1/CIP1) promoter showed that a GC-rich region located between positions -83 and -74, which contains a transforming growth factor beta-responsive element and one Sp1-binding site, is sufficient for the upregulation of p21(WAF1/CIP1) promoter by GGTI-298. Electrophoretic mobility shift assays showed a small increase in the amount of DNA-bound Sp1-Sp3 complexes. Furthermore, the analysis of Sp1 transcriptional activity in GGTI-298-treated cells by using GAL4-Sp1 chimera or Sp1-chloramphenicol acetyltransferase reporter revealed a significant increase in Sp1-mediated transcription. Moreover, GGTI-298 treatment also resulted in increased Sp1 and Sp3 phosphorylation. These results suggest that GGTI-298-mediated upregulation of p21(WAF1/CIP1) involves both an increase in the amount of DNA-bound Sp1-Sp3 and enhancement of Sp1 transcriptional activity. To identify the geranylgeranylated protein(s) involved in p21(WAF1/CIP1) transcriptional activation, we analyzed the effects of the small GTPases Rac1 and RhoA on p21(WAF1/CIP1) promoter activity. The dominant negative mutant of RhoA, but not Rac1, was able to activate p21(WAF1/CIP1). In contrast, constitutively active RhoA repressed p21(WAF1/CIP1). Accordingly, the ADP-ribosyl transferase C3, which specifically inhibits Rho proteins, enhanced the activity of p21(WAF1/CIP1). Taken together, these results suggest that one mechanism by which GGTI-298 upregulates p21(WAF1/CIP1) transcription is by preventing the small GTPase RhoA from repressing p21(WAF1/CIP1) induction.
Adnane
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Drug Discovery Program, H. Lee Moffitt Cancer Center, and Department of Biochemistry and Molecular Biology, University of South Florida, Tampa, Florida 33612, USA.
Bizouarn
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Qian
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Hamilton
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Sebti
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D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors*; D001549:Benzamides / Q000494:pharmacology*; D001665:Binding Sites / Q000235:genetics; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016193:G1 Phase / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics*; D005809:Genes, Regulator / Q000235:genetics*; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics; D010190:Pancreatic Neoplasms / Q000235:genetics; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000235:genetics*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000235:genetics*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000235:genetics*; D020742:rhoA GTP-Binding Protein
D001549:Benzamides; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C102521:GGTI 298; D009687:Nuclear Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D051705:Sp3 Transcription Factor; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein