Vesnarinone, a differentiation inducing drug, directly activates p21(waf1) gene promoter via Sp1 sites in a human salivary gland cancer cell line

Abstract

We previously demonstrated that a differentiation inducing drug, vesnarinone induced the growth arrest and p21(waf1) gene expression in a human salivary gland cancer cell line, TYS. In the present study, we investigated the mechanism of the induction of p21(waf1) gene by vesnarinone in TYS cells. We constructed several reporter plasmids containing the p21(waf1) promoter, and attempted to identify vesnarinone-responsive elements in the p21(waf1) promoter. By the luciferase reporter assay, we identified the minimal vesnarinone-responsive element in the p21(waf1) promoter at -124 to -61 relative to the transcription start site. Moreover, we demonstrated by electrophoretic mobility shift assay that Sp1 and Sp3 transcription factors bound to the vesnarinone-responsive element. Furthermore, we found that vesnarinone induced the histone hyperacetylation in TYS cells. These results suggest that vesnarinone directly activates p21(waf1) promoter via the activation of Sp1 and Sp3 transcription factors and the histone hyperacetylation in TYS cells.

Figure 1

Plasmid construction. pGL3-WWP is a reporter construct containing 2.3 kb p21^waf1 promoter sequence. pGL3-WWP-0.2, pWP124 and pWPdel-SmaI are 5′-deletion constructs of the p21^waf1 promoter. pGL3-WWP-0.2 contains 225 bp of p21^waf1 promoter sequence. pWP124 contains 134 bp and pWPdel-SmaI contains 70 bp of p21^waf1 promoter sequence.

Figure 2

Human p21^waf1 promoter sequence located between −215 bp and +19 bp. The transcription start site is indicated by the number 0 on the sequence. Sp1 binding sites tentatively termed Sp1-1, Sp1-2, Sp1-3, Sp1-4, Sp1-5 and Sp1-6 from the upstream are indicated by underlining and shown below the sequence. Sp1-A contains Sp1-1 and Sp1-2 sites, and Sp1-B contains Sp1-4, Sp1-5 and Sp1-6 sites. TATA box is also indicated by underlining.

Figure 3

Luciferase assay. TYS cells were seeded in 35 mm dishes in DMEM supplemented with 10% FCS. Twenty-four hours later, the cells were transfected in triplicate with 5 μg of the several reporter plasmids by use of the Superfect reagent. Fifteen hours after transfection, vesnarinone (50 μg ml⁻¹) was added, and 20 h later, cell lysates were collected. The luciferase activities of the cell lysates were measured with a Promega luciferase assay kit. Luciferase activities were normalized by the amount of protein in cell lysates. Data are shown as means (bars, s.d.), and are representative of three separate experiments with similar results.

Figure 4

Electrophoretic mobility-shift assay (A, B) and supershift assay (C). Nuclear extracts prepared from vesnarinone (50 μg ml⁻¹)- or DMSO-treated TYS cells were incubated with a ³²P-labelled Sp1-A probe or Sp1-B probe (A). Nuclear extracts from TYS cells after treatment with vesnarinone for 15, 30, 45 min and a labelled Sp1-A probe were incubated in the binding buffer (B). Protein samples were prepared from TYS cells after treatment with vesnarinone for 45 min. Polyclonal antibody against Sp1 and/or Sp3 was added to the binding reaction and incubated for 20 min at room temperature before addition of a labelled Sp1-A probe (C).

Figure 5

Histone acetylation in TYS cells induced by vesnarinone. Nuclear extracts were prepared from TYS cells after treatment with 50 μg ml⁻¹ vesnarinone or 10 μg ml⁻¹ TSA for 16 h. Protein samples were subjected to SDS–PAGE, transferred to nitrocellulose, and detected with an anti-acetylated Histone H3 antibody and Amersham ECL kit.