Increased polyamine intake inhibits age-associated alteration in global DNA methylation and 1,2-dimethylhydrazine-induced tumorigenesis

Abstract

Polyamines (spermine and spermidine) play many important roles in cellular function and are supplied from the intestinal lumen. We have shown that continuous high polyamine intake inhibits age-associated pathologies in mice. The mechanism by which polyamines elicit these effects was examined. Twenty-four week old Jc1:ICR male mice were fed one of three experimental chows containing different polyamine concentrations. Lifetime intake of high polyamine chow, which had a polyamine content approximately three times higher than regular chow, elevated polyamine concentrations in whole blood, suppressed age-associated increases in pro-inflammatory status, decreased age-associated pathological changes, inhibited age-associated global alteration in DNA methylation status and reduced the mortality in aged mice. Exogenous spermine augmented DNA methyltransferase activity in Jurkat and HT-29 cells and inhibited polyamine deficiency-induced global alteration in DNA methylation status in vitro. In addition, increased polyamine intake was associated with a decreased incidence of colon tumors in BALB/c mice after 1,2-demethylhydrazine administration; 12 mice (60%) in the low polyamine group developed tumors, compared with only 5 mice (25%) in the high polyamine group (Fisher's exact probability = 0.027, p = 0.025). However, increased polyamine intake accelerated the growth of established tumors; maximal tumor diameter in the Low and High groups was 3.85±0.90 mm and 5.50±1.93 mm, respectively (Mann-Whitney test, p = 0.039). Spermine seems to play important roles in inhibiting age-associated and polyamine-deficient induced abnormal gene methylation as well as pathological changes including tumorigenesis.

Figure 1. Metabolic pathway for polyamine synthesis, Dnmt activity and gene methylation.

SAM is a methyl donor, and dcSAM inhibits Dnmt activity. ODC = Ornithine decarboxylase; AdoMetDC = Adenosylmethionine decarboxylase; SAM = S-adenosylmethionine; dcSAM = Decarboxylate S-adenosylmethionine; Dnmt = DNA methyltransferase.

Figure 2. Survival curve.

The survival curve was created based on the results of 4 separate experiments. There were significant differences between the moderate and high polyamine chow groups (p = 0.010), and between the low and high polyamine groups (p = 0.040). No significant difference was found between the low and moderate polyamine groups (p = 0.613).

Figure 3. Blood polyamine levels, LFA-1 expression and methylation status.

3-a. Whole blood polyamine concentration. Polyamine concentrations in whole blood samples from young (24 weeks) and aged mice (80 weeks) were measured by HPLC. * Spermine concentrations in aged mice fed high polyamine chow were significantly higher than in the Low group or in young (24 weeks old) mice (p = 0.018 vs. Low, p = 0.006 vs. young mice) Bars indicate means and standard deviations. 3-b. Effect on CD11a expression. ^# The percentage of bright CD11a cells in aged mice fed high polyamine chow was significantly (p = 0.039) lower than in mice fed low polyamine chow, and was similar to that of young mice. Bars indicate means and standard deviations. 3-c. Methylation status of kidney genes. MS-AFLP array analysis using a mouse DNA array is shown. Control samples comprised a mixture of tissue from three young mice. Mean data of the log2 ratios of the values from 3 animals relative to the control samples are shown. Positive figures indicate increased demethylation of samples relative to the control, and negative figures indicate increased methylation of samples relative to the control. SPM = Spermine; SPD = Spermidine; Low polyamine = 80 weeks of mice fed low polyamine chow; High = 80 weeks of mice fed high polyamine chow. Young mice = 24 week-old mice.

Figure 4. Polyamine concentration, Dnmt activity and methylation status.

4-a. Polyamine concentration in cells under various conditions. DFMO treatment decreased, and spermine supplementation increased, concentrations of spermine and spermidine in Jurkat cells. Bars indicate means and standard deviations. 4-b. Effects of polyamine on DNA methyltransferase activity. * DFMO treatment significantly (64.20±28.60% of No treatment cells, p = 0.004) decreased, and ^# spermine supplementation increased (p = 0.004) methyltransferase activities in Jurkat cells. Bars indicate means and standard deviations. 4-c. Methylation status in Jurkat cells. MS-AFLP array analyses using a human DNA array are shown. DNA from untreated Jurkat cells was used as a control. The log2 ratios of values relative to the control samples are shown. Positive figures indicate increased demethylation of samples relative to the control, and negative figures indicate increased methylation of samples relative to the control. SPM = Spermine; SPD = Spermidine.

Figure 5. Effect of food polyamine on tumorigenesis.

Four-week old male mice (BALB/c) fed either the high polyamine or low polyamine chow were administered 20 mg/kg BW of 1,2-dimethylhydrazine (DMH) (2.0 mg/ml in PBS) subcutaneously for 12 consecutive weeks. Increased polyamine intake suppressed DMH-induced tumorigenesis, but appeared to enhance the growth of established tumors. Low = mice fed low polyamine chow; High = mice fed high polyamine chow.