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

Abstract

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.

FIG. 1

GGTI-298 upregulates p21^WAF1/CIP1 promoter activity in human pancreatic tumor cells, Panc-1 cells, in a p53-independent manner. Panc-1 cells were transfected with 4 μg of p21P, which contains the full-length sequence of p21 promoter, or p21PΔp53, which is lacking the p53 consensus site and 0.5 μg of pCMV-βgal as described in Materials and Methods. At 15 h posttransfection, cells were incubated with increasing doses of GGTI-298 for 36 h. The fold induction was calculated by dividing the luciferase activity values of samples treated with GGTI-298 by the activity of untreated control samples. The samples were normalized for transfection efficiency against β-galactosidase activity. Bars represent standard deviations. The data are representative of three independent experiments.

FIG. 2

Upregulation of p21^WAF1/CIP1 promoter by GGTI-298 is mediated through a region that contains a TβRE and Sp1-binding sites. Panc-1 cells were transfected with the indicated p21P deletion constructs. At 15 h posttransfection, cells were incubated in either medium alone or medium containing GGTI-298 (15 μM) for 36 h as described in Materials and Methods. The fold induction was calculated by dividing the luciferase activity values of samples treated with GGTI-298 by the activity of untreated control samples. The samples were normalized for transfection efficiency against β-galactosidase activity. Panc-1 cells were also transfected with pSRE to determine specificity of GGTI-298. Each error bar represents the average deviation for three independent experiments. The construct map was adapted from Datto et al. (6).

FIG. 3

Sp1- and TGF-β-responsive element at positions −83 to −78 is essential for GGTI-298-mediated upregulation of p21 promoter activity. Panc-1 cells were transfected with the indicated p21P mutant constructs. Starting at 15 h posttransfection, cells were incubated with GGTI-298 (15 μM) for 36 h. The fold induction was calculated by dividing the luciferase activity values of samples treated with GGTI-298 by the activity of untreated control samples. The samples were normalized for transfection efficiency against β-galactosidase activity. Each error bar represents the average deviation for three independent experiments. The construct map was adapted from Datto et al. (6). mut, mutation.

FIG. 4

Sp1 and Sp3 interact with GGTI-298-responsive region. Nuclear extracts from GGTI-298-treated or untreated Panc-1 cells were incubated with a ³²P-labeled probe corresponding to the sequence from −86 to −71 of the wt p21 promoter. Unlabeled wt or mutant competitors corresponding to the sequence from −86 to −71 of the wt p21 promoter and p21P93-S mut 2, respectively, were used. Polyclonal antibodies to either Sp1, Sp-3, or normal rabbit immunoglobulin G were included for supershift. Data are representative of two independent experiments.

FIG. 5

GGTI-298 upregulates Sp1-transcriptional activity. Panc-1 cells were cotransfected with 1 μg of GAL4-Sp1 constructs, 4 μg of G5BCAT, 2 μg of Sp1-CAT or E2F-CAT, and 0.5 μg of pCMV-βgal. At 15 h posttransfection, cells were incubated with GGTI-298 (15 μM) for 36 h as described in Materials and Methods. Samples were normalized for transfection efficiency against β-galactosidase activity and then assayed for CAT activity. Thin-layer chromatography plates were scanned with a PhosphorImager, and the percentages of acetylated and nonacetylated forms of chloramphenicol were determined. The fold induction was calculated by dividing the CAT activity values of samples treated with GGTI-298 by the activity of untreated control samples. Data are representative of three independent experiments.

FIG. 6

GGTI-298 mediates an increase in Sp1 and Sp3 phosphorylation. GGTI-298-treated and untreated Panc-1 cells were labeled with ortho[³²P]phosphate as described in Materials and Methods. Equal amounts of proteins were used for IP with Sp1 (1:200) and Sp3 (1:100) polyclonal antibodies, followed by analysis of the immunocomplexes by SDS–8% PAGE. The gel was fixed, dried, and exposed for autoradiography. Data are representative of two independent experiments.

FIG. 7

The small GTPase RhoA is an upstream effector of p21^WAF1/CIP1. (A) Panc-1 cells were cotransfected with 6 μg of p21^WAF1/CIP1 promoter, 4 μg of Rac1 or RhoA, and 0.5 μg of pCMVβ-gal expression vectors. Rac1-115I and RhoA-63L vectors express constitutively active GTPases. Rac1-17N and RhoA-19N vectors express dominant negative mutants. Aliquots of cell lysate were subjected to β-galactosidase and luciferase assays. (B) Panc-1 cells were transfected with p21^WAF1/CIP1 promoter, and at 15 h posttransfection cells were incubated in DMEM supplemented with 0.5% FBS for 24 h. Subsequently, cells were treated with C3 exoenzyme as described in Materials and Methods. Aliquots of cell lysate were analyzed for β-galactosidase and luciferase activities. Data are representative of at least three independent experiments.