Experiments relying on exogenous addition of polyamines to assay buffers or on the cellular uptake of polyamines added to cell culture medium do not always mimic the normal compartmentalization of polyamines synthesized naturally in the cell, or necessarily lead to the same biological outcome (6,35). For this reason, we have used strategies relying on the manipulation of the natural polyamine biosynthetic pathway to examine the consequences of elevated intracellular polyamines on histone acetylation in proliferating cells. Mammalian cells were retrovirally engineered to overexpress ODC, labeled with [3H] acetate, and acetylated histones examined by fluorography and Western analyses. Given the early work suggesting stimulatory effects of polyamines on HAT enzymes in crude extracts (28,29), we were not surprised to find that elevated levels of ODC promote acetylation of histones in the several immortalized epidermal and fibroblast cell lines tested (33). The ODC-mediated increase in acetylated histones was abrogated when cells were treated with DFMO, indicating that polyamines were specifically responsible for the observed effect on histone acetylation. Similar polyamine effects on histone acetylation were observed in G2/M-synchronized and asynchronously growing cell populations. Because deacetylation of lysine residues in histone tails by HDAC enzymes typically occurs very rapidly, cells were always labeled in the presence of 10 mM sodium butyrate, an effective HDAC inhibitor. Given that deacetylase activity was efficiently blocked in these experiments, the increased acetyl-ation observed in the ODC-overexpressing cells reflects a direct or indirect effect of polyamines specifically on HAT enzyme activity. Definitive assessment of the effects of polyamines on HDAC activity must await the development of specific, cell-permeable inhibitors of HAT enzymes.
The increase in acetylated histone H4 detected in cells constitutively overexpressing ODC was determined to be owing to an increase in hyperacetylated histone isoforms, rather than simply a greater overall level of monoacetylated histones (33). Hyperacetylated histones are found in nucleosomes associated with transcriptionally active chromatin. Therefore, our observation that ODC overexpression cooperates with histone deacetylase inhibitors to further enhance reporter gene expression beyond that promoted by ODC or inhibitor treatment alone (33) might be explained by the proportionally greater amounts of the more highly acetylated histone isoforms in these cells. The increased abundance of mono- and hyperacetylated histones in ODC-overexpress-ing cells necessitates that some proportion of nucleosomes comprise histones having a different combination of nonacetylated and acetylated lysine residues as compared with normal circumstances. These changes in the pattern of lysine acetylation may result in an altered binding affinity of nucleosomes for DNA, or altered interactions between neighboring nucleosomes, perhaps affecting nucleosomal positioning or access by factors critical for transcription and other cellular processes. In fact, highly specific patterns of acetylated lysine residues occur in histones that act as signals for the recruitment of proteins that facilitate the reconfiguration of chromatin associated with gene expression, DNA replication, repair, recombination, and differentiation. Thus the altered patterns of histone acetylation promoted by high intracellular concentrations of polyamines might be expected to cause aberrant function of one or more of these cellular programs. Indeed, inappropriate histone acetylation has been linked to both the loss of the differentiated phenotype and cell transformation (36,37).
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