Abstract
The proliferation of the rat intestinal mucosal IEC-6 cell line requires polyamines, whose synthesis is catalyzed by the enzyme ornithine decarboxylase (ODC). ODC inhibition leads to polyamine depletion, as well as inhibition of both cell proliferation and apoptosis by regulating gene expression. The NF-kappa B transcription factor regulates genes involved in apoptotic, immune, and inflammatory responses. In the present study we tested the hypothesis that NF-kappa B is activated following ODC inhibition. We found that the inhibition of ODC by alpha-difluoromethylornithine (DFMO) resulted in a approximately 50% decrease in intracellular putrescine levels within 1 h. NF-kappa B is activated by DFMO through the degradation of the inhibitory protein I kappa B alpha that sequesters NF-kappa B in the cytoplasm. The DFMO-induced NF-kappa B complexes contain the p65 and p50 members of the Rel protein family. DFMO-induced NF-kappa B activation was accompanied by the translocation of p65 from the cytoplasm into the nucleus. DFMO selectively inhibited a gene reporter construct dependent on the kappa B site present in the HLA-B7 gene. In contrast, DFMO had no effect on a gene reporter construct dependent on the kappa B site present in the interleukin-8 gene. Thus, we report that ODC inhibition activates the NF-kappa B transcription factor, which may mediate the altered physiological state of intestinal cells that occurs following polyamine depletion.
Highlights
The polyamines, spermidine and spermine, and their precursor putrescine are intimately required for cell growth and proliferation [1]
The DFMO-induced Nuclear factor B (NF-B) complexes at all times examined reflected the effects of ornithine decarboxylase (ODC) inhibition and blockage of polyamine synthesis because their induction was prevented by the addition of putrescine
To demonstrate directly that ODC inhibition by DFMO resulted in depletion of intracellular polyamine levels, the effect of DFMO treatment on the intracellular levels of putrescine was determined in intestinal epithelial cell (IEC)-6 cells
Summary
The polyamines, spermidine and spermine, and their precursor putrescine are intimately required for cell growth and proliferation [1]. Previous studies have established that inhibition of ODC activity in IEC-6 cells leads to alterations in gene expression [2]. Polyamine depletion by DFMO treatment in IEC-6 cells delays the onset of apoptosis by tumor necrosis factor-␣ and the DNA topoisomerase inhibitor camptothecin [3]. NF-B-inducing stimuli promote dissociation of the inactive NF-B/IB complexes via the serine phosphorylation and degradation of IB These events lead to the unmasking of the nuclear localization sequence of NF-B, thereby allowing NF-B to enter the nucleus and bind Bregulatory elements. ODC inhibition by DFMO depletes cellular polyamine levels and activates the NF-B transcription factor, which may repress the expression of important cellular genes in intestinal cells. The regulation of gene expression mediated by NF-B activation may manifest itself in alterations in cell physiology, such as the intrinsic resistance of polyamine-depleted cells to apoptosis
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