Polyamine Depletion Stabilizes p53 mRNA

In response to polyamine depletion, cellular levels of p53 protein are greatly increased, and the ability of p53 to bind specific DNA sequences is significantly activated (16,17,25,26). Because expression of the p53 gene is rapid and transient, it acts as a mediator, linking short-term stress signals to growth arrest or apoptosis by regulating the activation of specific genes. p53 is an ephemeral protein, and its half-life is approx 25-40 min in variety of cell types (17). In addition to transcriptional regulation, expression of the p 5 3 gene is primarily regulated at the posttranscriptional level. Increasing evidence suggests that phosphorylation plays a major role in the regulation of both the stability of p53 protein and its DNA-binding activity. It has been shown that phosphorylation of human p53 at Ser-15 and Ser-20 induces conformational changes in the NH2 terminus that disrupt Mdm2 binding and lead to its stabilization (16,17).

We have recently investigated the mechanisms of p53 gene expression by cellular polyamines in normal IECs (6,20). Polyamine depletion by treatment with DFMO dramatically increases the stability of p53 mRNA as measured by the mRNA half-life , but has no effect onp5 3 gene transcription in IEC-6 cells. As can be seen in Fig. 2, p53 mRNA levels in control cells declined rapidly after inhibition of gene transcription by addition of actinomycin D. The half-life of p53 mRNA in control cells was approx 45 min. However, the stability of p53 mRNA was dramatically increased by polyamine depletion with a half-life of longer than 18 h. Increased half-life of p53

Fig. 2. Cytoplasmic half-life studies of p53 mRNA from IEC-6 cells in the presence or absence of cellular polyamines. (A) Representative autoradiograms of Northern blots. Cells were untreated (a) or treated with DFMO alone (b) or DFMO plus SPD (c) for 6 d and then incubated with 5 mM actinomycin D/mL for indicated times. Total cellular RNA was isolated and p53 mRNA levels were assayed by Northern blot analysis using a p53 complimentary DNA probe. Loading of RNA was monitored by hybridization to labeled GAPDH probe. (B) Percent of p53 mRNA remaining in IEC-6 cells described in A. Values are means ± SE of data from three separate experiments, and relative levels of p53 mRNA were corrected for RNA loading as measured by densitometry of GAPDH.

Fig. 2. Cytoplasmic half-life studies of p53 mRNA from IEC-6 cells in the presence or absence of cellular polyamines. (A) Representative autoradiograms of Northern blots. Cells were untreated (a) or treated with DFMO alone (b) or DFMO plus SPD (c) for 6 d and then incubated with 5 mM actinomycin D/mL for indicated times. Total cellular RNA was isolated and p53 mRNA levels were assayed by Northern blot analysis using a p53 complimentary DNA probe. Loading of RNA was monitored by hybridization to labeled GAPDH probe. (B) Percent of p53 mRNA remaining in IEC-6 cells described in A. Values are means ± SE of data from three separate experiments, and relative levels of p53 mRNA were corrected for RNA loading as measured by densitometry of GAPDH.

mRNA was prevented when spermidine was given together with DFMO. The half-life of p53 mRNA in cells treated with DFMO plus spermidine was approx 48 min, similar to that of controls (without DFMO). In contrast, inhibition of polyamine synthesis did not increase p53 gene transcription as measured by using nuclear run-on transcription assays (20). There were no significant differences in the rate of p53 gene transcription between control cells and cells exposed to DFMO in the presence or absence of sper-midine for 6 d. These findings clearly indicate that polyamines regulate the p53 gene expression posttranscriptionally in IECs, and that depletion of cellular polyamines induces p53 mRNA levels primarily through the increase in its stability.

Posttranscriptional regulation, especially modulation of mRNA stability, has been shown to play an important role in gene expression (18,19). The turnover rate of a given mRNA can be determined by interactions of tra n ¿-acting factors with specific c/'s-element(s) located within 3^-untranslated regions (3$ UTR) (27-29). Many labile mRNAs including those that encode transcription factors contain AU-rich elements in their 3$ UTR. The presence of a reiterated pentamer (5$-AUUUA-3 $) in many AUrich elements is associated with rapid mRNA turnover and translation attenuation (27). Deletion of the AU-rich element region enhances mRNA stability and insertion of the region into the 3 $ UTR of a normally stable globin mRNA significantly destabilizes it (27-29). Several AUUUA-binding proteins have been identified in different cell types, although the mechanisms of how these proteins affect mRNA turnover are unclear. It is unclear at present that an AU-rich element region in the p53 3 $ UTR is critical for the stabilization of p53 mRNA after polyamine depletion in IECs.

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