Adnane J, Bizouarn F A, Qian Y, Hamilton A D, Sebti S M
Drug Discovery Program, H. Lee Moffitt Cancer Center, and Department of Biochemistry and Molecular Biology, University of South Florida, Tampa, Florida 33612, USA.
Mol Cell Biol. 1998 Dec;18(12):6962-70. doi: 10.1128/MCB.18.12.6962.
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.
我们最近报道，香叶基香叶基转移酶I抑制剂GGTI-298可使人类肿瘤细胞停滞于细胞周期的G1期，并增加细胞周期蛋白依赖性激酶抑制剂p21（WAF1/CIP1）的蛋白质和RNA水平。在此，我们表明GGTI-298在转录水平发挥作用，在人胰腺癌细胞系Panc-1中诱导p21（WAF1/CIP1）的表达。p21（WAF1/CIP1）启动子的这种上调具有选择性，因为GGTI-298抑制血清反应元件和E2F介导的转录。对p21（WAF1/CIP1）启动子的功能分析表明，位于-83至-74位之间的富含GC的区域足以使GGTI-298上调p21（WAF1/CIP1）启动子，该区域包含一个转化生长因子β反应元件和一个Sp1结合位点。电泳迁移率变动分析显示，与DNA结合的Sp1-Sp3复合物数量略有增加。此外，通过使用GAL4-Sp1嵌合体或Sp1-氯霉素乙酰转移酶报告基因对GGTI-298处理的细胞中的Sp1转录活性进行分析，结果显示Sp1介导的转录显著增加。此外，GGTI-298处理还导致Sp1和Sp3磷酸化增加。这些结果表明，GGTI-298介导的p21（WAF1/CIP1）上调涉及与DNA结合的Sp1-Sp3数量的增加以及Sp1转录活性的增强。为了鉴定参与p21（WAF1/CIP1）转录激活的香叶基香叶基化蛋白，我们分析了小GTP酶Rac1和RhoA对p21（WAF1/CIP1）启动子活性的影响。RhoA的显性负突变体而非Rac1能够激活p21（WAF1/CIP1）。相反，组成型活性RhoA抑制p21（WAF1/CIP1）。因此，特异性抑制Rho蛋白的ADP-核糖基转移酶C3增强了p21（WAF1/CIP1）的活性。综上所述，这些结果表明GGTI-298上调p21（WAF1/CIP1）转录的一种机制是通过阻止小GTP酶RhoA抑制p21（WAF1/CIP1）的诱导。
