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. 2003 Jun 24;100(13):7859-64.
doi: 10.1073/pnas.1332465100. Epub 2003 Jun 16.

Pronounced reduction in adenoma recurrence associated with aspirin use and a polymorphism in the ornithine decarboxylase gene

Maria Elena Martinez  1 , Thomas G O'Brien, Kimberly E Fultz, Naveen Babbar, Hagit Yerushalmi, Ning Qu, Yongjun Guo, David Boorman, Janine Einspahr, David S Alberts, Eugene W Gerner
Affiliations

Pronounced reduction in adenoma recurrence associated with aspirin use and a polymorphism in the ornithine decarboxylase gene

Maria Elena Martinez et al. Proc Natl Acad Sci U S A. .

Abstract

Most sporadic colon adenomas acquire mutations in the adenomatous polyposis coli gene (APC) and show defects in APC-dependent signaling. APC influences the expression of several genes, including the c-myc oncogene and its antagonist Mad1. Ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, is a transcriptional target of c-myc and a modifier of APC-dependent tumorigenesis. A single-nucleotide polymorphism exists in intron 1 of the human ODC gene, which lies between two myc-binding domains. This region is known to affect ODC transcription, but no data exist on the relationship of this polymorphism to risk of colorectal neoplasia in humans. We show that individuals homozygous for the minor ODC A-allele who reported using aspirin are approximately 0. 10 times as likely to have an adenoma recurrence as non-aspirin users homozygous for the major G-allele. Mad1 selectively suppressed the activity of the ODC promoter containing the A-allele, but not the G-allele, in a human colon cancer-derived cell line (HT29). Aspirin (>or=10 microM) did not affect ODC allele-specific promoter activity but did activate polyamine catabolism and lower polyamine content in HT29 cells. We propose that the ODC polymorphism and aspirin act independently to reduce the risk of adenoma recurrence by suppressing synthesis and activating catabolism, respectively, of colonic mucosal polyamines. These findings confirm the hypothesis that the ODC polymorphism is a genetic marker for colon cancer risk, and support the use of ODC inhibitors and aspirin, or other nonsteroidal antiinflammatory drugs (NSAIDs), in combination as a strategy for colon cancer prevention VSports手机版. .

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Figures

Scheme 1.
Scheme 1.
Fig. 1.
Fig. 1.
Odds ratios for adenoma recurrence as a function of ODC genotype and aspirin use. Results of logistic regression models that control for age, gender, and number of colonoscopies are shown (95% confidence intervals). Individuals homozygous for the G-allele and who reported no aspirin use are the reference (Ref) group. The number of individuals who recurred over the total in each group among non-aspirin users are 137/266 (51.5%), 100/182 (55.0%), and 13/31 (41.9%) for GG, AG, and AA variants, respectively; the corresponding figures among aspirin users are 55/116 (47.4%), 34/82 (41.5%), and 2/11 (18.2%).
Fig. 2.
Fig. 2.
A single-nucleotide polymorphism (SNP) in the ODC promoter and its significance for APC-dependent ODC promoter activity. (A) ODC gene structure. There are three consensus-binding sites for the c-myc:Max and Mad1:Max heterodimers within the ODC gene. These sites, called E-boxes, have the consensus sequence CACGTG. E-box (1) is located in the 5′ flanking region of the gene, and E-box (2) and E-box (3) are separated by 28 nt in the first intron. The G/A SNP (*) is positioned between the two E-boxes in intron 1 at +316. Also marked in this diagram are the nearby CpG sequences. (B) Effect of wild-type APC expression on the level of three E-box transcription factors. HT29-APC and control HT29-β-gal cells were treated with 300 μM ZnCl2 for 5 h. Whole-cell lysates from untreated and treated cells were run on an SDS/PAGE gel and Western blots were performed for c-myc, Mad, and Max, as described in Methods. (C) Effect of wild-type APC expression on allelespecific ODC promoter activity. HT29-APC (open bars) and HT29-β-gal (filled bars) cells were transfected with either pGL3-ODC/A or pGL3-ODC/G, as described in Methods. Transfected cultures were then treated with or without 300 μM ZnCl2 for an additional 24 h and harvested, and lysates were analyzed for luciferase activity. Results are presented as the ratios of mean values ± standard deviations of triplicate measurements from cultures treated with ZnCl2 divided by those from similarly transfected cells not treated with ZnCl2. The result is an average of three experiments. Bars indicate standard deviations. *, Statistically significant effects (P < 0.05). (D) Effect of Mad1 expression on allele-specific ODC promoter activity. By using HT29 cells, either pGL3-ODC/A or pGL3-ODC/G was cotransfected with pcDNA3.1 or pcDNA-Mad1 in a molar ratio of 3:1. The cells were harvested after 48 h, and lysates were analyzed for luciferase activity. Experiments were performed in triplicate. Bars indicate standard deviation. *, Statistically significant effects (P < 0.05).
Fig. 3.
Fig. 3.
Aspirin induces SSAT promoter, RNA, and enzyme activity in HT29 cells. (A) SSAT promoter activity. HT29 cells were transfected with reporter SSAT-Luc along with pCMV-β-gal plasmid. Relative luciferase units (RLU) were calculated after normalizing to the protein and β-gal activities in the cell lysates. Fold induction was calculated after dividing the RLU of sample by the RLU of vehicle. Bars indicate standard deviation. *, Statistically significant difference (P < 0.05) compared with vehicle. (B) SSAT RNA. HT29 cells were seeded and grown for 24 h. The cells were then treated with indicated amounts of aspirin for 48 h and harvested, and total RNA was extracted. Total RNA was analyzed by probing for SSAT and GAPDH. Fold induction was calculated by dividing normalized sample values to the GAPDH control. Bars indicate standard deviation. *, Statistically significant difference (P < 0.05) compared with vehicle. (C) SSAT enzyme activity. HT29 cells were grown overnight and then treated with either various concentrations of aspirin or its vehicle for 24 h. Cells were harvested and SSAT enzyme activity was measured as described in Methods. The results are an average from three different experiments. Bars indicate standard deviation. *, Statistically significant difference (P < 0.05) compared with vehicle.
Fig. 4.
Fig. 4.
Decrease in polyamine levels in HT29 cells caused by aspirin. Cells were treated for 48 h with either 20 or 100μM aspirin or its vehicle (ethanol). Polyamine levels were normalized to the protein in the samples. Fold changes were then calculated after dividing the polyamine level in the sample by the polyamine level in the vehicle control and plotted. Bars indicate standard deviations. *, Statistically significant difference (P < 0.05) compared with vehicle.
Fig. 5.
Fig. 5.
Model depicting actions of ODC polymorphism and aspirin on polyamine metabolism, and the consequences for adenoma recurrence. Before acquisition of somatic APC mutations, colonic cells in individuals with the minor A-allele would have reduced polyamine synthesis due to selective suppression of ODC promoter activity by Mad1. Aspirin would act, in an ODC allele-independent manner, to reduce further polyamine levels by activating the transcription of SSAT, thereby increasing polyamine catabolism and export. Cancer risk would be directly related to colonic mucosal polyamine contents, being decreased by decreased polyamine levels.
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