p21(WAF1/CIP1) is upregulated by the geranylgeranyltransferase I inhibitor GGTI-298 through a transforming growth factor β- and Sp1- responsive element: Involvement of the small GTPase RhoA

Jalila Adnane, Francisco A. Bizouarn, Yimin Qian, Andrew Hamilton, Saïd M. Sebti

Research output: Contribution to journalArticle

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 β-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.

Original languageEnglish (US)
Pages (from-to)6962-6970
Number of pages9
JournalMolecular and Cellular Biology
Volume18
Issue number12
StatePublished - Dec 1998

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Monomeric GTP-Binding Proteins
Transforming Growth Factors
Up-Regulation
GC Rich Sequence
Cyclin-Dependent Kinase Inhibitor p21
Cell Cycle Proteins
Chloramphenicol O-Acetyltransferase
GGTI 298
geranylgeranyltransferase type-I
DNA
G1 Phase
Electrophoretic Mobility Shift Assay
Transferases
Adenosine Diphosphate
Transcriptional Activation
Proteins
Binding Sites
Phosphorylation
RNA
Cell Line

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

Cite this

p21(WAF1/CIP1) is upregulated by the geranylgeranyltransferase I inhibitor GGTI-298 through a transforming growth factor β- and Sp1- responsive element : Involvement of the small GTPase RhoA. / Adnane, Jalila; Bizouarn, Francisco A.; Qian, Yimin; Hamilton, Andrew; Sebti, Saïd M.

In: Molecular and Cellular Biology, Vol. 18, No. 12, 12.1998, p. 6962-6970.

Research output: Contribution to journalArticle

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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 β-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.",
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T1 - p21(WAF1/CIP1) is upregulated by the geranylgeranyltransferase I inhibitor GGTI-298 through a transforming growth factor β- and Sp1- responsive element

T2 - Involvement of the small GTPase RhoA

AU - Adnane, Jalila

AU - Bizouarn, Francisco A.

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AU - Hamilton, Andrew

AU - Sebti, Saïd M.

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N2 - 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 β-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.

AB - 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 β-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.

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