Abstract
We first demonstrated that long-term increased polyamine (spermine, spermidine, putrescine) intake elevated blood spermine levels in mice and humans, and lifelong consumption of polyamine-rich chow inhibited aging-associated increase in aberrant DNA methylation, inhibited aging-associated pathological changes, and extend lifespan of mouse. Because gene methylation status is closely associated with aging-associated conditions and polyamine metabolism is closely associated with regulation of gene methylation, we investigated the effects of extracellular spermine supplementation on substrate concentrations and enzyme activities involved in gene methylation. Jurkat cells and human mammary epithelial cells were cultured with spermine and/or D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase. Spermine supplementation inhibited enzymatic activities of adenosylmethionine decarboxylase in both cells. The ratio of decarboxylated S-adenosylmethionine to S-adenosyl-L-methionine increased by DFMO and decreased by spermine. In Jurkat cells cultured with DFMO, the protein levels of DNA methyltransferases (DNMTs) 1, 3A and 3B were not changed, however the activity of the three enzymes markedly decreased. The protein levels of these enzymes were not changed by addition of spermine, DNMT 3A and especially 3B were activated. We show that changes in polyamine metabolism dramatically affect substrate concentrations and activities of enzymes involved in gene methylation.
Highlights
Polyamines are linear aliphatic hydrocarbons with three or more primary amino groups
We have shown that aberrant methylation status induced by inhibiting ornithine decarboxylase (ODC) was almost reversed by spermine supplementation [24], and that life-long consumption of high-polyamine chow by mice inhibited aging-associated changes in methylation status of the entire genome, inhibited aging associated pathological changes, and extended lifespan [14,16]
Cells were collected for use in experiments after sufficient passages resulted in a large enough number of cells, and incubated for 72 h in the following conditions: 1. control culture (Jurkat: RPMI-1640 + 10% human inactivated serum; human m2a.mRmesauryltsepithelial cells (HMEpCs): serum-free medium); 2. culture mixed with D,L-alpha-difluoromethylornithine (DFMO) (Amine Pharma Institute, Chiba, Japan), an irreversible inhibitor of ODC; 3. culture with spermine (Sigma-Aldrich Japan, Tokyo); and 4. culture with DFMO and spermine
Summary
Polyamines are linear aliphatic hydrocarbons with three or more primary amino groups. AdoMetDC activity in Jurkat cells (50.81 ± 41.52 pmol/mg protein/min) cultured with DFMO markedly increased in comparison with that in control cells (i.e., cultured without DFMO or spermine) (1.56 ± 0.77 pmol/mg protein/min) (p < 0.001) (Figure 4a). The dcSAM concentration significantly increased in Jurkat cells cultured with DFMO (7.12 ± 0.74 pM) (p = 0.001 vs control culture (0.34 ± 0.12 pM) and significantly decreased in response to spermine addition in the absence (0.043 ± 0.074 pM) (p = 0.031 vs control culture (0.34 ± 0.12 pM) or presence (dcSAM concentration below the detection limit) (p < 0.001 vs DFMO culture (7.12 ± 0.74 pM) of DFMO (Figure 4c). AdoMetDC activity markedly decreased in spermine-treated cells in the absence (0.28 ± 0.045 pmol/mg protein/min) (p < 0.001 vs control culture) and presence of DFMO (0.57 ± 0.13 nmol/mg protein/min) (p = 0.002 vs DFMO culture) (Figure 4e).
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