WTH3 is a direct target of the p53 protein

Abstract

Previous results showed that overexpression of the WTH3 gene in multidrug resistance (MDR) cells reduced MDR1 gene expression and converted their resistance to sensitivity to various anticancer drugs. The WTH3 gene promoter was found to be differentially regulated in paired MDR vs non-MDR MCF7 cells owing to epigenetic modifications and transcription factor modulations. To understand further the mechanisms that govern WTH3's differential expression, we uncovered a p53-binding site in its promoter, which indicated that WTH3 could be regulated by the p53 gene. This hypothesis was then tested by different strategies. The resulting data revealed that (1) the WTH3 promoter was upregulated by the p53 transgene in diverse host cells; (2) there was a correlation between WTH3 expression levels and p53 gene status in a cell line panel; (3) a WTH3 promoter region was directly targeted by the p53 protein in vitro and in vivo. In addition, overexpression of the WTH3 gene promoted the apoptotic phenotype in host cells. On the basis of these findings, we believe that the negative role played by the WTH3 gene in MDR development is through its proapoptotic potential that is regulated by multiple mechanisms at the transcription level, and one of these mechanisms is linked to the p53 gene.

Figure 1

Results obtained from luciferase assays where pGL3 or pGL/WTH3P was cotransfected with pcDNA3.1, pcDNA/P53 or pcDNA/P53R249S into (A) MCF7/WT and (B) HEK293 cells. Relative luciferase activities driven by the WTH3 promoter under the influence of the empty vector, wild-type and mutated p53 were compared.

Figure 2

(A) Results obtained from SQRT-PCR to estimate endogenous WTH3 gene expressions in 11 cell lines with defined p53-gene status. (B) Western blot analysis of WTH3 proteins in MCF7/AdrR vs MCF7/WT and MES-SA/D × 5 vs MES-SA using WTH3 (1 : 200 dilution) and Rab6 (1 : 2000 dilution) antibodies. In addition, MDR1 detected by the MDR1 antibody (1 : 30 dilution) served as one of the controls. Proteins and their molecular weights are indicated on the right and left. Identical SDS-PAGE gels with the same amount of protein (100 μg/lane) were loaded. Three gels were used for Western blot by three antibodies; the fourth gel was stained with Coomassie blue for protein concentration normalisation.

Figure 3

Results obtained from SQRT-PCR to estimate endogenous WTH3 gene expressions in Hela and MCF7/AdrR cells, which were influenced by the p53 transgene. (A) The two cell lines were either transfected with pcDNA3.1 or pcDNA/P53, where β-actin served as the quantitative control. (B) Quantitative comparison of the results presented in (A). The empty and black columns represent WTH3 expression levels under the influence of the empty vector or the p53 transgene, respectively.

Figure 4

Luciferase assay results for the WTH3 promoter deletion mutants under the influence of the p53 gene in HEK293 cells. The empty and black columns represent relative luciferase activity driven by the empty vector (pGL3), wild-type WTH3 and four deleted promoter mutants (pGL/WTH3P-d1, -d2, -d3 and -d4), which were either influenced by the empty vector (pcDNA3.1) or the p53 transgene.

Figure 5

(A) Results obtained from EMSA and super-EMSA assays where P49 was used as the probe that was interacted with the purified p53 protein. Lane 1 contains the labelled probe. Lane 2 contains the labelled probe and 50 ng of p53 protein. The solid arrow indicates the DNA/p53 complexes. Lane 3 contains the labelled probe, 50 ng of p53 protein and the anti-p53 antibody. The empty arrow indicates the antibody shifted DNA/p53 complexes. Lane 4 contains the labelled probe, 50 ng of p53 protein and 100 times excess of cold P49. (B) Results obtained from EMSA and super-EMSA assays where the P49 probe and nuclear extracts obtained from MCF7/AdrR and MCF7/WT were used. Lane 1 contains the labelled probe. Lane 2 contains the labelled probe and 10 μg of nuclear extract prepared from MCF7/WT cells. The solid arrow indicates the DNA/p53 complexes. Lane 3 contains the labelled probe, 10 μg of MCF7/WT nuclear extract and the anti-p53 antibody. The empty arrow indicates the antibody shifted DNA/p53 complexes. Lane 4 contains the labelled probe, 10 μg of nuclear extract prepared from MCF7/AdrR cells. Lane 5 contains the labelled probe, 10 μg of MCF7/AdrR nuclear extract and the anti-p53 antibody. (C) Chromatin immunoprecipitation assay using MCF7/WT cells treated with 1 μM DOX. p53–DNA complexes were captured with anti-p53 antibody. Anti-HA antibody was used as the negative control. PCR products representing the WTH3, p21 (positive control) and albumin promoter (negative control) sequences are noted.

Figure 6

(A and B) results obtained from DAPI staining assays using Hela cells. In (A) and (B) Hela cells contain pcDNA3.1 and pcDNA/WTH3, respectively. Cells were stained with DAPI, and the stained nuclei were visualised under a fluorescent microscope (magnification, × 400). Arrows indicate apoptotic nuclear condensation. (C–F) results obtained from TUNEL assays using Hela and HEK293 cells as hosts. (C and D) HEK293 cells were transfected with pcDNA3.1 and pcDNA/WTH3, respectively, for 30 h. (E and F) Hela cells were transfected with pcDNA3.1 and pcDNA/WTH3, respectively, for 18 h. DNA fragmentation in the apoptotic cells induced by the WTH3 gene were stained with a brown colour as compare to the control cells containing the empty vector, which were blue in colour. (G–I) results generated by flow cytometry assays using HEK293 cells. (G) Cells served as vehicle control. (H and I) cells were transfected with pcDNA/3.1 and pcDNA/WTH3, respectively. The cells were then stained with PI and the intercellular fluorescence was measured by a flow cytometer. The sub-G1 DNA content in each group, which represented apoptotic cells, is indicated by a percentage.