Increased p53 Causes Growth Inhibition in Polyamine Deficient Cells

The following three studies provide evidence showing that increased accumulation of p53 protein plays a critical role in the process of growth inhibition after polyamine depletion in normal IECs. In the first study, the time course of p53 gene expression and cell growth were examined after polyamine-deficient cells were exposed to exogenous spermidine (6,20). IEC-6 cells were initially treated with DFMO for 4 d and then exposed to spermidine. The changes in both p 5 3 gene expression and cellular DNA synthesis were measured at various times after spermidine was added to the medium containing DFMO. Depletion of cellular polyamines by administration of DFMO for 4 d increased the steady-state levels of p53 mRNA and protein. When these polyamine-deficient cells were exposed to exogenous spermidine, prevention of increased p53 mRNA and protein preceded the beginning of cellular DNA synthesis as measured by [3H]-thymidine incorporation. The significant decreases in p53 mRNA and protein levels were observed 12 h after exposure to exogenous spermidine, whereas the increased rate of DNA synthesis occurred at 24 h after administration of spermidine. These findings indicate that the restoration of cell growth by exogenous spermidine in polyamine-deficient cells is preceded by a decrease in p53 gene expression.

In the second study, the effect of inhibition of p53 expression by using p53 antisense oligodeoxyribonucleotides on cell growth was examined in polyamine-deficient IEC-6 cells (20). Cells were grown in the presence of DFMO for 4 d and DNA synthesis was assayed by [3H]-thymidine incorporation 48 h after administration of different concentrations of p53 antisense or sense oligomers. Exposure of polyamine-deficient cells to p53 antisense oligomers significantly increased DNA synthesis in the presence of DFMO. Treatment with p53 antisense oligomers also inhibited p53 protein expres-

Fig. 3. Effects of inhibition of nucleophosmin (NPM) expression by treatment with the siRNA-targeting NPM mRNA-coding region (siNPM) on levels of p53 protein and its transcriptional activity in polyamine-deficient cells. (A) Representative immunoblots for NPM and p53 proteins. Cells were grown in the culture containing DFMO for 4 d and transfected with either control siRNA (C-siRNA) or siNPM by lipofectamine technique. Cells were harvested 48 h after transfection in the presence of DFMO, and levels of NPM and p53 proteins were measured by Western blot analysis. Equal loading of proteins was monitored by actin immunoblotting. (B) Changes in p53 transcriptional activity as measured by p21 promoter activity in cells described in (A). Cells were exposed to DFMO for 4 d and then transfected with p21-promoter luciferase reporter plasmid and either siNPM or C-siRNA. After 48 h of incubation in the presence of DFMO, transfected cells were harvested and assayed for luciferase activity. Data of p21 promoter activity were normalized by b-galactosidase activity from cotransfection plasmid pRSV b-galactosidase. Values are means ± SE of data from six dishes. *, + p < 0.05 compared with controls and DFMO-treated cells transfected with C-siRNA, respectively.

Fig. 3. Effects of inhibition of nucleophosmin (NPM) expression by treatment with the siRNA-targeting NPM mRNA-coding region (siNPM) on levels of p53 protein and its transcriptional activity in polyamine-deficient cells. (A) Representative immunoblots for NPM and p53 proteins. Cells were grown in the culture containing DFMO for 4 d and transfected with either control siRNA (C-siRNA) or siNPM by lipofectamine technique. Cells were harvested 48 h after transfection in the presence of DFMO, and levels of NPM and p53 proteins were measured by Western blot analysis. Equal loading of proteins was monitored by actin immunoblotting. (B) Changes in p53 transcriptional activity as measured by p21 promoter activity in cells described in (A). Cells were exposed to DFMO for 4 d and then transfected with p21-promoter luciferase reporter plasmid and either siNPM or C-siRNA. After 48 h of incubation in the presence of DFMO, transfected cells were harvested and assayed for luciferase activity. Data of p21 promoter activity were normalized by b-galactosidase activity from cotransfection plasmid pRSV b-galactosidase. Values are means ± SE of data from six dishes. *, + p < 0.05 compared with controls and DFMO-treated cells transfected with C-siRNA, respectively.

sion in DFMO-treated cells. When DFMO-treated cells were exposed to 2 mM a nt i-sense oligomers for 48 h, increased p53 protein levels were prevented as measured by Western blot analysis and immunohistochemical staining. Treatment with sense p53 oligomers at the same concentrations, however, showed no significant effects on DNA synthesis and p53 protein expression. These results provide direct evidence that increased expression of the p53 gene plays a critical role in growth inhibition of normal intestinal epithelial cells after polyamine depletion.

In the third study, the influence of decreased p53 stability and transcriptional activity by inhibiting NPM expression on cell proliferation was examined in polyamine-deficient IEC-6 cells. Increased NPM in DFMO-treated cells was associated with a significant decrease in cell growth, which was completely prevented by exogenous putrescine (32). In studies using NPM siRNA, cells were initially grown in the medium containing DFMO for 4 d, and then transfected with NPM siRNA or C-siRNA. Cell numbers were assayed 48 h after transfection in the presence of DFMO and minimal putrescine (0.5 mM). Decreased levels of p53 through inhibition of NPM by transfec-

tion with NPM siRNA significantly promoted cell proliferation in polyamine-deficient cells. The results suggest, for the first time, that NPM/p53 signaling is involved in ne gative control of normal IEC growth after polyamine depletion.

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