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Mutant p53 in MDA-MB-231 breast cancer cells is stabilized by elevated phospholipase D activity and contributes to survival signals generated by phospholipase D
L Hui, Y Zheng, Y Yan, J Bargonetti and D A Foster
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Figure 1.
Elevated expression of p53 in MDA-MB-231 cells is dependent upon PLD. (a) The level of p53 protein in MCF7 and MDA-MB-231 (231) cells was determined by Western blot analysis as described previously (Hui et al., 2004). To control for loading, blots were re-probed with an antiactin antibody. (b) MDA-MB-231 cells (231 cells) were transfected with either control (Con) GAPDH or PLD2 siRNA (Ambion) and PLD activity was determined using the transphosphatidylation reaction as described previously (Hui et al., 2004). The PLD activity in the cells treated with the PLD2 siRNA was normalized to the control, which was given a value of one. Error bars represent the standard deviation for duplicate samples. (c) Cells transfected with siRNA in B were also analysed for PLD2 protein levels by Western blot analysis. (d) MDA-MB-231 cells were transfected with either control (Con) GAPDH or PLD2 siRNA and the level of p53 was determined 72 h later as in (a). Experiments in (a, b, c and d) are representative of experiments repeated at least two times. MDA-MB-231 and MCF7 cells were obtained from the American Type Culture Collection and were maintained as described previously (Chen et al., 2005). Transfection of siRNA was performed using LipofectAMINE 2000TM reagent (Invitrogen, Chicago, IL, USA) according to the vendor's instructions. Monoclonal antibodies raised against p53 (pAb240 and pAb421), which recognize DNA-binding and C-terminal regions of p53 (Bargonetti et al., 1993), were used for Western blot detection of p53. Antibodies against PLD2 and actin were from Upstate Biotechnology, Charlotesville, VA, USA and Santa Cruz Biotechnology, Santa Cruz, CA, USA, respectively.
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Figure 2.
Elevated expression of p53 in MDA-MB-231 cells is dependent on MAP kinase and independent of mTOR. (a) MDA-MB-231 cells (231 cells) were transfected with either control (Con) GAPDH or PLD2 siRNA as in Figure 1. The levels of phosphorylated MAP kinase (P-MAPK), MAP kinase (MAPK), phosphorylated MEK (P-MEK) and actin were then determined by Western blot analysis. (b) MDA-MB-231 cells were treated with either solvent (dimethyl sulfoxide (DMSO)) or the MAP kinase inhibitor U0126 (Cell Signaling Technology, Danvers, MA, USA) (20 
M) for 1 h. The levels of p53, phosphorylated MAP kinase (P-MAPK) and MAPK were then determined by Western blot analysis. (c) MDA-MB-231 cells were treated with either DMSO solvent (0.1%) or rapamycin (Sigma, St Louis, MO, USA) (Rap) (20
M) for 1 h. The levels of p53, phosphorylated S6 kinase (S6K) and S6K were then determined by Western blot analysis. Experiments shown in (a, b and c) are representative of experiments repeated at least two times. Antibodies against MAP kinase, phosphorylated MAP kinase, S6 kinase, phosphorylated S6 kinase and phosphorylated MEK were from Cell Signaling Technology.
Figure 3.
Suppression of PLD or MAP kinase signaling increases the turnover of p53 and increases the association of p53 and HDM2. (a) MCF7 and MDA-MB-231 cells were treated with cyclohexamide (Calbiochem, San Diego, CA, USA) (CHX) (80 g/ml) for the indicated times. Cells were then harvested and p53 levels were determined by Western blot analysis. In the lower panel is a representation of the data in the upper panel from densitometer tracings of the autoradiograms in the upper panel. (b) MDA-MB-231 cells were treated with either U0126 (Calbiochem) (20
M) or DMSO control for 1 h. The cells were treated with cyclohexamide and p53 levels were determined as in (a). (c) MDA-MB-231 cells were transfected with either PLD2 or GAPDH control siRNA as in Figure 1. Seventy-two hours later the cells were treated with cyclohexamide and p53 levels were determined as in (a). (d) MDA-MB-231 cells were transfected with either PLD2 or GAPDH control siRNA as in Figure 1. Seventy-two hours later, lysates were immunoprecipited (IP) with an anti-p53 antibody (Cell Signaling Technology). The immunoprecipitates were then subjected to Western blot analysis (IB) with either HDM2 (Santa Cruz Biotechnology) or p53 (Bargonetti et al., 1993) antibodies as indicated. The strategy was then reversed and lysates were IP with HDM2 followed by Western blot with p53 and HDM2 antibodies as described previously (Hui et al., 2004). (e) MDA-MB-231 cells were treated with either U0126 or DMSO control for 1 h. The cells were then lysed and the lysates were IP with an anti-p53 antibody and the immunoprecipitates were then subjected to Western blot analysis (IB) with either HDM2 or p53 antibodies as in (a). (f) MDA-MB-231 cells were transfected with either PLD2 or GAPDH control siRNA as in (a) and HDM2 levels were determined by Western blot. (g) MDA-MB-231 cells were treated with either U0126 or DMSO control for 1 h and HDM2 levels were determined as in (f). Experiments shown are representative of experiments repeated at least two times.
Figure 4.
Survival signals generated by PLD is dependent upon mutant p53. (a) MDA-MB-231 cells were plated in Dulbecco's modified Eagle's medium (DMEM) with 10% serum for 24 h. The cells were then transfected with either control GAPDH or PLD2 siRNA. Twenty-four hours later, the cells were shifted to fresh media containing 0% serum for 48 h, at which time the percentage of non-viable cells was determined by trypan blue exclusion as described previously (Hui et al., 2004, Hui et al., 2005). At this time, cells were also examined for the level of cleaved PARP (Cl PARP) and PLD2 protein using Western blot analysis as described previously (Hui et al., 2004, Hui et al., 2005). Blots were stripped and reprobed with an antibody to actin to control for loading. (b) MDA-MB-231 cells were plated in DMEM with 10% serum for 24 h. The cells were then shifted to fresh media containing 0% serum for 16 h in the presence of either U0126 (20
M) or the solvent DMSO, at which time the percentage of non-viable cells and the level of cleaved PARP (Cl PARP) was determined as in (a). The effect of U0126 on MAP kinase phosphorylation is also shown. (c) MDA-MB-231 cells were plated in DMEM with 10% serum for 24 h. The cells were then transfected with either control GAPDH or p53 siRNA. Twenty-four hours later, the cells were shifted to fresh media containing 0% serum for 48 h, at which time the percentage of non-viable cells and the level of cleaved PARP (Cl PARP) was determined as in (a and b). The effect of p53 siRNA on p53 protein levels is also shown. The experiments shown in (a, b and c) are representative of at least two independent experiments.
