Fig 1.
Semi-quantitative RT-PCR analysis of mRNAs specific for MCAK and GAPDH (A), western analysis of protein levels of MCAK, p53, p21WAF1/Cip1, and β-actin in continuously growing HCT116 (p53+/+) and HCT116 (p53−/−) cells (B), and western blot analysis of individual protein levels in HCT116 (p53+/+) cells following treatment with the control scramble RNAi or Stealth RNAi for p53 (C), and in MCF7 cells stably transfected with control shRNA vector or shp53 vector (D). RT-PCR was performed using gene-specific primers as described in Materials and methods. To ensure that the same amount of RNA was being used, the total RNA concentration was normalized to that of GAPDH as the message of a housekeeping gene. HCT116 (p53+/+) cells were treated with each RNAi for 48 h. Western blot analysis was performed using ECL Western Blotting Kit as described in Section 2. A representative study is shown and two additional experiments yielded similar results.
Fig 2.
Diagrammatic representation of the transcription factor binding sites in the MCAK promoter-reporter constructs (A), comparison of the promoter activities among pGL2-basic, pGL2-530, pGL2-530/mutated p53-motif, and pGL2-320 constructs in HCT116 (p53+/+) cells (B), and effect of ectopically expressed p53 on the promoter activities of pGL2-530, pGL2-530/mutated p53-motif, and pGL2-320 constructs in p53-deficient HCT116 cells (C). The cells were transiently transfected or cotransfected with individual the promoter-reporter constructs with wild-type p53-expression vector (pCMV-Neo/p53) or empty-vector (pCMV-Neo). The pSV-β-galactosidase control vector was cotransfected in each experiment to correct for variations in transfection efficiency. The luciferase and β-galactosidase activities were measured as described in Materials and methods. Each value is expressed as the mean ± SD (n = 3). *Statistical significance was defined as p<0.05 compared to the control.
Fig 3.
Nucleotide sequence and putative transcription factor binding sites (A), and mutational analysis of the two p53-REs (p53-RE1 at −173/−166 and p53-RE2 at −245/−238) and two Sp1 binding motifs (GC1 at −93/−84 and GC2 at −119/−110) (B) in the human MCAK promoter (pGL2-320-Luc). The nucleotide sequence of the MCAK promoter region from −266 to +54 was analyzed and the positions of the putative transcription factor binding site are underlined, and the transcription start site is designated as +1. The translation start codon ATG is shaded light gray. The individual site-directed mutations were introduced within the pGL2-320-Luc vector. Transient transfection with each mutation construct was performed in HCT116 (p53+/+) and HCT116 (p53−/−) cells, and then luciferase activity was determined. Each value is expressed as the mean ± SD (n = 3). *Statistical significance was defined as p<0.05 compared to the control.
Fig 4.
ChIP analyses to detect the suppressive effect of p53 overexpression on the in vivo association of Sp1 with the MCAK promoter (A) and western blot analyses of individual protein levels in HCT116 (p53−/−) cells (B). The cells were transiently transfected with wild-type p53-expression vector (pCMV-Neo/p53) or empty-vector (pCMV-Neo). ChIP assays were performed using a ChIP Assay Kit. Formaldehyde-cross-linked chromatin was prepared from the cells and then immunoprecipitated with anti-Sp1 antibody or normal mouse IgG. The recovered immunoprecipitates were analyzed by PCR with specific primers to amplify the 320-bp MCAK promoter region (−266/+54) possessing two p53-REs (p53-RE1 at −173/−166 and p53-RE2 at −245/−238) as well as two GC-motifs (GC1 at −93/−84 and GC2 at −119/−110) using the SacI-forward primer (5'-AGGA-GCTCAGTCAAGTTTCTAATCTG-3') and the BglII-reverse primer-2 (5’-AGAGATCTCGGAGA-GTCAGCAAGGAAGAG-3’). The 320-bp PCR products were resolved on a 1.5% agarose gel electrophoresis and are indicated by arrows. Western blot analyses were performed as described in Materials and methods. A representative study is shown and two additional experiments yielded similar results.
Fig 5.
Differential effect of Sp1 and p53 on the MCAK core promoter (pGL2-320-Luc) activity (A), the pG5-5×(GC)-Luc activity (B), and pGL2-mdm2 promoter (pGL-mdm2-Luc) activity (C). One hundred nanograms of individual promoter-reporter constructs (pGL2-320-Luc, pG5-5×(GC)-Luc, and pGL2-mdm2-Luc) were cotransfected with pCAGGS-Sp1, pCAGGS-wt-p53, or pCAGGS control empty vector into HCT116 (p53-/-) cells, and then the luciferase activity was determined. Each value is expressed as the mean ± SD (n = 3). *Statistical significance was defined as p<0.05 compared to the control (100 ng of pCAGGS). To analyze the expression level of MCAK, Sp1, and β-actin proteins in the cells after transient transfection, western blot analyses were performed as described in Materials and methods. A representative study is shown and two additional experiments yielded similar results.
Fig 6.
Effect of mithramycin A on the Sp1-dependent activation of MCAK promoter activity of the proximal core MCAK promoter pGL2-320 (A) and the endogenous levels of MCAK, Sp1, p53, and β-actin (B), and the arbitrary densitometric units of the individual proteins normalized to those of β-actin (C) in HEK-293 cells. HEK 293 cells were transiently transfected with either 0.2 μg of the promoter-reporter construct pGL2-320 (-266/+54) or equivalent amount of the control vector pGL2-basic. After transfection for 24 h, the cells were treated with various concentrations of mithramycin A (0, 100, and 500 nM). The luciferase activity was measured 48 h after transfection and normalized for the amount of protein. The mean ± SD of three independent experiments performed in duplicate is shown. *Statistical significance was defined as p<0.05 compared to the control. Under the same condition, equivalent amounts of cell lysates were analyzed for the endogenous levels of MCAK, Sp1, p53, p21WAF1/Cip1, and β-actin by western blotting. A representative study is shown and two additional experiments yielded similar results.
Fig 7.
Effect of nutlin-3a on the endogenous levels of MCAK, Sp1, p53, p21WAF1/Cip1, and β-actin (B) in HCT116 (p53+/+) and HCT116 (p53−/−) cells.
The cells were treated with various concentrations of nutlin-3a (1, 5, and 10 μM). After treatment for 48 h, the cells were harvested and cell lysates were prepared. Equivalent amounts of cell lysates were analyzed for endogenous levels of MCAK, Sp1, p53, p21WAF1/Cip1, and β-actin by western blotting. A representative study is shown and two additional experiments yielded similar results. The mean ± SD of three independent experiments performed in duplicate is shown. *Statistical significance was defined as p<0.05 compared to the control. The arbitrary densitometric units obtained for the MCAK protein were normalized to those of β-actin in HCT116 (p53+/+) and HCT116 (p53−/−) cells.
Fig 8.
Differential effect of wild-type p53 or tumor-derived p53 mutants on the MCAK core promoter (pGL2-320-Luc) activity (A), and the endogenous levels of MCAK, p53, Sp1, p21WAF1/Cip1, and β-actin (B), and the arbitrary densitometric units of the individual proteins normalized to those of β-actin (C) in HCT116 (p53−/−) cells. The promoter-reporter construct pGL2-320 was cotransfected into HCT116 (p53−/−) cells with empty-vector (pCMV-Neo), or each expression construct of wt-p53 (pCMV-Neo/p53wt), mutant p53V143A (pCMV-Neo/p53V143A), mutant p53R248W (pCMV-Neo/p53R248W), and mutant p53R273H (pCMV-Neo/p53 R273H), and then luciferase activity was determined. Each value is expressed as the mean ± SD (n = 3). *Statistical significance was defined as p<0.05 compared to the control. Under these conditions, equivalent amounts of cell lysates were analyzed for the endogenous levels of individual proteins by western blotting. A representative study is shown and two additional experiments yielded similar results.
Fig 9.
Cytoplasmic and nuclear localizations of Sp1, wt-p53, and p53 mutants (p53V143A, p53R248W, and p53R273H) in HCT116 cells (A), and ectopic p53 expression-mediated down-regulation of MCAK in HeLa and SW480 (B). After HCT116 (p53-/-) cells were transfected with pCMV-Neo, pCMV-wt-p53, pCMV-p53V143A, pCMV-p53R248W, and pCMV-p53R273H, the cells were fractionated into cytosolic and nuclear fractions. Western blot analyses were performed to assess the subcellular localization of MCAK, p-p53 (Ser-15), p53, p-Sp1 (Thr-453), Sp1, PARP, CDK4, and β-actin. CDK4 and β-actin were used as cytoplasmic markers, and PARP was used as the nuclear marker. Both HeLa and SW480 cells transfected with pCMV-Neo and pCMV-wt-p53, respectively, were subjected to western blot analysis of MCAK, p53, Sp1, and β-actin. A representative experiment is shown, and two additional experiments yielded similar results.