Polyamine Depletion Associated With Increased p53 Gene Expression

Considerable interest over the past several years has been devoted to the mechanisms of negative growth control. In the small intestinal mucosa, for example, mitotic activity is confined to the crypt region and must be finely tuned to the rapid rate of loss of mature enterocytes from the villous tip. The o reti c al ly, mucosal epithelial cell turnover is controlled by both positive and negative control mechanisms, with stimulation or suppression of cell renewal mediated through growth-promoting or growth-inhibiting genes. Although growth-promoting genes have been extensively studied in vivo and in vitro, studies to elucidate the role and significance of negative growth control in the regulation of cell growth have recently started.

The p53 gene encodes for a nuclear phosphoprotein, which was originally discovered as a cellular protein bound to the SV40 T antigen in transformed cells. p53 is present in low concentrations in normal cells and has a half-life of 6-20 min (16,17). Expression of the p53 gene is highly regulated by the cell according to its state of growth. Steady-state levels of p53 mRNA are significantly increased in certain growth conditions and in cells transformed by a variety of means, including viral infection, chemical treatment, and transfection of oncogenes (18,19). Inactivation of the p53 gene occurs in more than half of all human tumors, implying that expression of the p53 gene has an important physiological role in the control of the cell cycle and that loss of this gene represents a fundamental step in the pathogenesis of cancer (16,17). Thi s action of p53 has been ascribed to its ability to induce expression of a cellular gene WAF1/CIP1 that encodes a 21-kDa inhibitor of Gj cyclin-dependent kinases (17,19). In addition, there is increasing evidence that the p53 protein also induces apoptosis (16). Activation of the p53 gene expression induces growth arrest, apoptosis, or both.

The observation that p53 protein can suppress cell growth suggests that p53 may play an important role in the control of the cell cycle, which regulates the orderly flow of the cell in and out of the Gj phase during normal cell proliferation (16-20). p53 functions as a transcriptional factor and is a cell-cycle control protein because progression from the Gj to S phase is often blocked in cells expressing high p53 protein levels (17). Mercer and colleagues (21) demonstrated that induction of p53 expression by trans-fecting a conditional p53 expression plasmid inhibits cell-cycle progression and that, when growth-arrested cells are stimulated to proliferate, induction of p53 expression inhibits G0/Gj progression into the S phase. p53 also is required for the Gj arrest caused by g-ray exposure (16,17).

The first evidence showing the role of the p53 gene expression in growth inhibition after polyamine depletion is from our observations (6,20) and others (22,23) in normal IECs. Using cultured IEC-6 cells, we have made the unique observation that inhibition of polyamine synthesis with DFMO significantly increases p53 gene expression, which is associated with an increase in Gj phase growth arrest but not apoptosis (6). Increases in p53 after polyamine depletion were associated with increases in other cell-cycle inhibitors, including p21Waf1/ClP1 and p27Kip1. In control cells, steady-state levels of p53 mRNA were present at 4 d and then almost completely disappeared at 6 d, with minimal expression at 12 d after plating. Depletion of cellular polyamine through DFMO treatment significantly increased expression of the p53 gene (Fig. 1). This increase in mRNA levels for the p53 gene was noted at 4 d and remained elevated at 12 d after exposure to DFMO. Maximum increases in p53 mRNA levels occurred between 6 and 12 d after addition of DFMO and were more than 10 times that of control values. The increased levels of p53 mRNA in polyamine-deficient cells were paralleled by increases in p53 protein. Spermidine given together with DFMO completely prevented the increased

Fig. 1. Expression of p53 in control IEC-6 cells and cells exposed to 5 mM a-difluoromethyl-ornithine (DFMO) or DFMO plus spermidine (SPD). (A) Representative autoradiograms from control cells and cells exposed to DFMO or DFMO + SPD for 4, 6 and 12 d. Total cellular RNA was isolated and mRNA levels were examined by Northern blot analysis using p53 and Rb gene complimentary DNA probes. (B) Representative immunoblots for p53 protein as measured by Western blot analysis. p53 protein was identified by probing nitrocellulose with a specific anti-p53 antibody. Three experiments were performed that showed similar results.

Fig. 1. Expression of p53 in control IEC-6 cells and cells exposed to 5 mM a-difluoromethyl-ornithine (DFMO) or DFMO plus spermidine (SPD). (A) Representative autoradiograms from control cells and cells exposed to DFMO or DFMO + SPD for 4, 6 and 12 d. Total cellular RNA was isolated and mRNA levels were examined by Northern blot analysis using p53 and Rb gene complimentary DNA probes. (B) Representative immunoblots for p53 protein as measured by Western blot analysis. p53 protein was identified by probing nitrocellulose with a specific anti-p53 antibody. Three experiments were performed that showed similar results.

expression of the p5 3 gene. The concentrations of p53 mRNA and protein in cells treated with DFMO plus spermidine were indistinguishable from those in cells grown in control cultures. The effect of polyamines on p53 gene expression is specific because polyamine depletion did not induce expression of the Rb gene in IEC-6 cells. The se results suggest that p53 is involved in the regulation of intestinal mucosal growth by polyamines.

Consistent with our findings, Kramer and colleagues (17,22) have reported that exposure of human melanoma cells (MALME-3M cell) to a polyamine analog, WN11-diethylnorspermdine (DENSPM), not only decreases cellular polyamines but also increases p53 and p21 gene expression. DENSPM is known to deplete polyamine pools by both inhibiting biosynthetic enzymes and potently inducing the polyamine catabolic enzyme spermidine/spermine N^acetyltransferase. Treatment with DENSPM increases wild-type p53 (approx 10-fold at maximum), which is concomitant with an increase in p21 in MALME-3M cells. Another cyclin-dependent kinase inhibitor, p27, and cyclin D1 increase slightly, whereas proliferating cell nuclear antigen and p130 remain unchanged. Induction of p21 protein is paralleled by an increase in its mRNAb ut induction of p53 protein is not, suggesting that cellular polyamines dictate transcrip-tional activation of the p21 gene and posttranscriptional regulation of the p53 gene. Inconsistent with observations in IEC-6 cells, polyamine depletion by DENSPM in MALME cells also causes an increase in hypophosphorylated Rb protein.

In addition, polyamines also regulate extracellular signal-regulated kinase (ERK) activity in IEC-6 cells (23). Polyamine depletion results in an approx 50% increase in ERK-1 activity, and supplementation with putrescine restores the basal level of ERK-1 activity. Polyamine depletion has only a marginal effect on the level of ERK-1 protein. On the other hand, polyamine depletion increases ERK-2 activity 150% compared with control in IEC-6 cells. This increase in ERK-2 is largely prevented by addition of putrescine to DFMO-treated cells. Interestingly, polyamine depletion significantly reduced the level of ERK-2 protein. Similarly, the activity of the c-Jun NH2-terminal kinase is elevated on polyamine depletion and remains elevated in the presence of putrescine. The level of JNK protein is low in polyamine-depleted cells and returns to normal with the addition of putrescine. Inhibition of polyamine synthesis also induces STAT3 tyrosine phosphorylation in IEC-6 cells (24). However, the exact roles of polyamine depletion-modulated ERK and c-Jun NH2-terminal kinase activities and STAT3 phosphorylation in regulation of p53 and p21 expression remain to be elucidated.

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