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United States Department of Agriculture

Agricultural Research Service

2009 Annual Report

1a.Objectives (from AD-416)
LAB:Vitamins and Carcinogenesis To determine the complex roles the 'one-carbon nutrients', (methionine, choline and the B-vitamins, folate, B12, B6, and B2), as well as components of the diet that are one-carbon antagonists (such as alcohol) play in modifying metabolic and genetic pathways that lead to human cancer.

To define how the mechanistic knowledge acquired through objective #1 should be used to modify dietary habits, nutritional supplementation, and other nutritional interventions in order to prevent cancer. New advances made by this laboratory can thus be translated into public health initiatives that effectively reduce the burden of cancer in our society.

To define the biochemical, molecular and pathophysiologic processes that underlie the apparent effects of vitamin D and calcium in the modulation of cancer development, and to examine how genetic background and environmental factors further impact these effects.

1b.Approach (from AD-416)
LAB:Vitamins and Carcinogenesis Mechanistic questions will largely be examined in studies utilizing cell cultures and animal models. We have several cell lines derived from normal human colonic epithelial cells. The availability of one-carbon nutrients for these cells can easily be manipulated to examine the consequences of limited, or supplemental levels of nutrient availability. A variety of mouse models will also be used to examine the consequences of limited nutrient availability, including genetically engineered animals who either have a predisposition towards colon cancer or a polymorphism in a folate-dependent enzyme. Studies conducted in human volunteers, in which they undergo folate depletion for several weeks, will also be used to examine mechanistic questions, whereas intervention trials, where people at enhanced risk of colon cancer are randomly chosen to receive folate or placebo, will be used to translate this mechanistic work into answers regarding the possible utility of folate in the prevention of cancer.

3.Progress Report

We have numerous projects designed to define: which 1-carbon nutrients modify cancer risk and which underlying cellular pathways mediate these effects, how other factors such as genetic background, age, and alcohol intake modulate these relationships. The knowledge of these relationships gained from our work may ultimately be exploited in the prevention of cancer. Our mechanistic studies have included those conducted in cultured colon and breast cells, into which the common human C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene has been inserted. These studies demonstrated that folate availability and the polymorphism interact to determine genomic DNA methylation. Another interesting model, still developing, involves the implantation of murine colonocytes that express luciferase and which have been grown under various nutritional conditions, into nude mice and monitoring tumorigenesis in vivo. The genetic construct has been inserted into colonocytes and we are now optimizing growth conditions for these cells. Our animal studies are also designed to study mechanisms by which 1-carbon nutrients modify cancer risk. In collaboration with others we have constructed a mouse that has a conditional knockout of the MTHFR gene, and we are presently characterizing the biochemical phenotype of this animal. We have also completed a large study using mice to examine whether age and alcoholism act synergistically with folate depletion to enhance cellular pathways leading to colonic carcinogenesis. Lastly, we have initiated studies to utilize a very unique rodent model of colon carcinogenesis in order to define the paradoxical cancer-promoting effect of excessive folic acid administration. Our mechanistic work in both cells and animals continues to point towards the Wnt signaling pathway as a critical one in mediating the effects of folate on cancer. Signaling through this pathway, which is pro-carcinogenic, is enhanced by depletion of 1-carbon nutrients. We just completed two collaborative clinical studies in which ten volunteers took folic acid supplements for two months, and ten more were subjected to a folate depletion diet for two months. At baseline and at the 1 and 2 month time points we took colonic biopsies to examine gene expression, and select molecular anomalies relevant to cancer. Interesting alterations in genes relevant to 1-carbon metabolism and carcinogenesis were observed. Another clinical study examined polymorphisms in the DNA repair genes of urban Puerto Ricans in order to determine whether any of these polymorphisms alone, or in conjunction with 1-carbon vitamin status, determined uracil incorporation into blood DNA, a potentially mutagenic event. Four polymorphisms were identified as significant determinants of uracil levels. Also, in collaboration with the Epidemiology Group at the National Cancer Institute, we measured tissue folate concentrations in 800 colonic biopsies from a case-control study of normal subjects and those with colonic neoplasms. These data demonstrate that high folate levels in the tissue are associated with protection against the adenomas of most concern: the ‘high-risk adenoma.’

1. Genetic variations in DNA repair determine uracil levels in human DNA. Uracil is a nucleotide that normally does not appear as one of the four ‘building blocks’ of DNA, so its insertion into DNA can lead to an increased risk of cancer. Cells contain specific repair mechanisms that excise uracil from DNA and replace it with the appropriate nucleotide and these repair mechanisms depend on adequate folate availability. We examined a population of approximately 500 urban Puerto Ricans to determine whether common genetic variants in the repair mechanism predict uracil levels in the blood DNA from these individuals. We found 4 common variants that were associated with altered uracil levels although folate status in this population did not alter the relationships that we detected. These common genetic variants may help us predict who is at higher risk of cancer and also assist in determining whether vitamin status in such individuals is an important co-factor in determining risk.

2. Effects of folate intake on molecular markers in the colon. We completed two clinical studies and examined how:.
1)folic acid supplementation, and.
2)folate depletion alters the expression of genes in the human colon and how it alters certain other select molecular events that are relevant to the risk of developing cancer. These are unique studies that provide important insights into the mechanisms by which either folate excess or inadequacy modulate the risk of colorectal cancer in humans.

3. Colonic folate levels in humans and the risk of precancerous polyps. In collaboration with the epidemiology group at University of Minnesota we examined how the concentration of folate in the lining of the colon correlates with the risk of developing precancerous polyps of the colon. We examined a population of 800 individuals who underwent colonoscopy and we found that higher levels of folate in the colon were associated with substantial protection only against those particular types of polyps that are most likely to go on to develop into cancer. This underscores the importance of folate in cancer prevention and provides insights into the stage of cancer development in which folate plays a role.

4. Creation of a genetically engineered mouse for folate metabolism. In collaboration with the Harvard Center for Genomics and Genetics, we completed construction of a novel mouse whose methylenetetrahydrofolate reductase (MTHFR) gene has been deleted in a ‘conditional’ manner. The conditional feature enables us to selectively delete the gene in those tissues where it is desirable to remove enzyme activity. This type of mouse has never been previously constructed and it offers unique opportunities to determine the mechanism by which folate modulates the risk of cancer, and further, how common genetic variants in the MTHFR enzyme modulate the risk of cancer, as they are known to do.

5. Alcohol: effects on human chromatin structure. Alcohol, which inhibits the metabolism of 1-carbon vitamins, is a significant risk factor for colon cancer. We have investigated the effect of alcohol on modifications of a highly specialized protein, histone H3 modifications, that binds to DNA and is involved in determining the expression of certain cancer-related genes, such as the p16 gene. Alcohol at low concentrations produced sizeble changes in histone H3 and occurred in parallel with increased p16 expression. This is the first report of how alcohol modifies these specialized DNA-binding proteins and how that alters the expression of genes that are pivotal in cancer development, and the paper has been submitted for publication.

Review Publications
Sauer, J.A., Mason, J.B., Choi, S. 2009. Too much folate – a risk factor for cancer and cardiovascular disease?. Current Opinion in Clinical Nutrition and Metabolic Care. 12:30-36.

Mason, J.B., Choi, S., Liu, Z. 2008. Other 1-carbon micronutrients and age modulate the effects of folate on colorectal carcinogenesis. Nutrition Reviews. 66(Supplement 1):S15-7.

Chanson, A., Parnell, L.D., Ciappio, E.D., Liu, Z., Crott, J.W., Tucker, K., Mason, J.B. 2009. Polymorphisms in uracil-processing genes, but not one-carbon nutrients, are associated with altered DNA uracil concentrations in an urban Puerto Rican population. American Journal of Clinical Nutrition. 89:1927-1936.

Mason, J.B. 2009. Folate, cancer risk, and the greek god, Proteus: a tale of two chameleons. Nutrition Reviews. 67(4): 206-212.

Devos, L., Chanson, A., Liu, Z., Ciappo, E., Parnell, L.D., Mason, J.B., Tucker, K., Crott, J.W. 2008. Associations between single nucleotide polymorphisms in folate uptake and metabolizing genes with blood folate, homocysteine and DNA uracil concentrations. American Journal of Clinical Nutrition. 88(4):1149-1158.

Choi, S., Friso, S. 2009. Nutrients and DNA Methylation. In: Choi, S.W., Friso, S., editors. Nutrients and Epigenetics. Boca Raton, FL: CRC Press, Taylor & Francis Group. p.105-119.

Friso, S., Lamon-Fava, S., Jang, H., Schaefer, E., Corrocher, R., Choi, S. 2007. Estrogen replacement therapy reduces total plasma homocysteine and concurrently enhances genomic dna methylation in postmenopausal women. British Journal of Nutrition. 97(4):617-621.

Last Modified: 11/30/2015
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