Progress 10/01/99 to 09/30/04
Outputs
The etiologies of folate-related pathologies have both a genetic and nutritional component. Folate is a water-soluble B-vitamin, and folate-mediated one-carbon metabolism is necessary for the synthesis of DNA precursors and for SAM biosynthesis, and these pathways are inhibited by folate deficiency. Disruptions in folate metabolism lead to pathologies that are specific for an individual tissue including megaloblastic anemia, neural tube defects, cancers and nonketotic hyperglycinaemia. Disruption of folate metabolism can occur not only as a result of folate deficiency, but also due to other physiological, genetic and nutritional factors including iron status. Current research is focused on the development and study of mouse models to investigate the role of altered folic acid metabolism in folate-related disease. We study these models to determine how folate status can influence disease processes on various genetic backgrounds. These approaches will lead to new
strategies to diagnose and treat folate-related pathologies. Several interesting mouse models have been generated and preliminary investigations have been conducted. First we have knocked out the murine gene that encodes serine hydroxymethyltransferase (cSHMT). This gene regulates the supply of folate-activated single carbon units to the dTMP and SAM biosynthesis pathways. Mice lacking the cSHMT gene are viable and fertile, but may be conditionally lethal to females because they are underrepresented in all litters. Mice lacking the cSHMT gene do not accumulate fat stores; no other unusual phenotypes have been observed. Mice with increased cSHMT expression exhibit seizures and become infertile after 5 weeks of age. A full metabolite and biochemical characterization of these strains is now underway. We have also generated mice with increased expression of methenyltetrahydrofolate synthetase (MTHFS). Our laboratory has recently demonstrated that MTHFS catalyzes the
catacholamine-dependent degradation of folate and thereby regulates intracellular folate concentrations. We have shown that MTHFS is unregulated in animal tumors, which are known to exhibit increased rates of folate catabolism and folate deficiency. We have generated mice that can express the MTHFS cDNA in a tissue specific manner and thereby create localized cellular folate deficiency. These mice are being used to investigate the role of folate catabolism in (A) regulating cellular folate concentrations in a whole animal, (B) inducing folate deficiency independent of dietary supply, and (C) influencing folate metabolic pathways and disease processes. These studies are now underway.
Impacts
Epidemiological studies implicate impaired folate metabolism in several pathologies including neural tube defects, cardiovascular disease and cancer. Folate metabolism can be impaired by folate deficiency and/or genetic mutations and polymorphisms and affects genome wide methylation, gene expression and genome stability. The mechanisms that regulate intracellular folate concentrations and metabolism are largely unknown and understudied. Individuals have been identified who display folate deficiency despite having adequate folate intake, indicating that cellular folate deficiency or impairments in folate metabolism can occur even in the absence of dietary folate deficiency. For example, cancer patients and murine cancer models display increased rates of folate catabolism, which may lead to, and account for observed localized tissue folate deficiencies in the absence of whole-body folate deficiency. Genetic background has been shown to affect folate metabolism, folate
requirements, and risk for folate-associated disease. Therefore, elucidating the mechanisms that regulate systemic folate concentrations and one-carbon metabolism, as well as the folate content of individual tissues, is critical to understanding the complex relationships among folate status, folate metabolism and disease in genetically diverse populations.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
The studies outlined in this proposal seek to elucidate the role of key genes that regulate folate metabolism in folate-associated pathologies. Folate metabolism is required for the synthesis of DNA precursors, DNA methylation and numerous other cellular methylation reactions. Disruption of folate metabolism, either by folate deficiency or due to genetic polymorphisms, results in identifiable pathologies including anemias, neural tube defects and certain cancers. Our laboratory has identified several genes, including serine hydroxymethyltransferase, heavy chain ferritin and 10-formyltetrahydrofolate dehydrogenase, that regulate folate status and folate metabolism. Using internal ribosome entry sequences (IRES) and Cre/lox technologies, we have created a DNA construct that enables cell-specific and temporal deletion of these genes. To date, we have generated a stable line that contains the modified cSHMT gene and ferritin gene. Homozygous deletion of the cSHMT gene
results in a distorted sex bias of 3 females to one male due to high rates of male lethality. Homozygous deletion is lethal. Metabolic tracer studies indicate that these mice will have accelerated homocysteine remethylation capacity, increased DNA methylation density, impaired thymidine biosynthesis and exhibit pathologies associated with these biochemical markers. We are now studying the role of this gene on folate-associated pathologies (neural tube defects, colon cancer) as described in the proposal. We are in the progress of generating constructs that will be used to alter intracellular folate concentration as occurs during pregnancy and tumor progression. We have cloned the murine heavy chain ferritin gene as well as the murine 10-formyltetrahydrofolate dehydrogenase gene and generating mouse lines that express these cDNAs. Both of these new mouse strains are expected to exhibit symptomatic folate deficiency independent of folate intake. The role of tissue specific folate
deficiency on folate-associated related pathologies will be performed as described above.
Impacts
Impairments of folate metabolism are associated with many pathologies including cancer, birth defects and anemia. These studies will determine the role of critical genes that influence or mediate the effects of folate metabolism on these pathologies. From these studies, it is anticipated that new preventative strategies for folate-related disease will emerge.
Publications
- Stover, P. J. and Garza, C. 2002. Bringing Individuality to Public Health Recommendations. J. Nutr. 132, 2476S-2480S.
- Stover, P. J. and Garza, C. 2002. Molecular and genetic considerations for long-term nutrition interventions. Asia Pacific J. Clin. Nutr. In press.
- Herbig, A. K., Chiang, E.-P, Lee, L.-R., Hills, J. L., Shane, B. and Stover, P. J. 2002. Cytoplasmic Serine Hydroxymethyltransferase mediates Competition Between Folate-Dependent Deoxyribonucleotide and S-Adenosylmethionine Biosyntheses. J. Biol. Chem. 277, 38381-38390.
- Zanetti, K. A. and Stover, P. J. 2002. Pyridoxal Phosphate Inhibits Dynamic Subunit Interchange among Cytoplasmic Serine Hydroxymethyltransferase Tetramers. J. Biol. Chem. In press.
- Stover, P. J. 2002. Nutrition and Developmental Biology. Annales Nestle 60 (1), 1-11.
- Stover, P. J. 2002. Regulation of Folate Metabolism by Iron. in Folate and Human Development Massaro, EL and Rogers, JM, EDS. Humana Press New Jersey pp. 241-262.
- Anguera, M, and Stover, P. J. 2002. Rapid Affinity Purification of the T-protein from Rabbit Liver. In Chemistry and Biology of Pteridines and Folates (Milstein, s, Kapatos, G., Levine, RA, and Shane, B., eds.) Kluwer Academic Publishers, Boston. pp. 485-490.
- Herbig, A. K., Girgis, S. and Stover, P. J. 2002. Effects of Cellular Glycine Concentration on Human Cell Proliferation in Culture. In Chemistry and Biology of Pteridines and Folates (Milstein, s, Kapatos, G., Levine, RA, and Shane, B., eds.) Kluwer Academic Publishers, Boston. pp. 491-494.
- Allen, LH, Bentley, ME, Donovan, SM, Ney, DM and Stover, PJ 2002. Securing the Future of Nutritional Sciences Through Integrative Graduate Education J. Nutr. 132, 779-784.
- Garza, C and Stover, P. 2002. The role of science in identifying common ground in the GMO debate. From: IEH workshop on Gentically Modified Foods for Human Health and Nutrition.
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Progress 01/01/01 to 12/31/01
Outputs
The studies outlined in this proposal seek to elucidate the role of key genes that regulate folate metabolism in folate-associated pathologies. Folate metabolism is required for the synthesis of DNA precursors, DNA methylation and numerous other cellular methylation reactions. Disruption of folate metabolism, either by folate deficiency or due to genetic polymorphisms, results in identifiable pathologies including anemias, neural tube defects and certain cancers. Our laboratory has identified several genes, including serine hydroxymethyltransferase, heavy chain ferritin and 10-formyktetrahydrofolate dehydrogenase, that regulate folate status and folate metabolism. Using internal ribosome entry sequences (IRES) and Cre/lox technologies, we have created a DNA construct that enables cell-specific and temporal deletion of the cytoplasmic serine hydroxymethyltransferase (cSHMT) gene. We have generated over 100 progeny that contains the modified cSHMT gene. Homozygous
deletion of the cSHMT gene results in a distorted sex bias of 3 females to one male due to high rates of male lethality. Homozygous deletion is lethal. Metabolic tracer studies indicate that these mice will have accelerated homocysteine remethylation capacity, increased DNA methylation density, impaired thymidine biosynthesis and exhibit pathologies associated with these biochemical markers. We are now studying the role of this gene on folate-associated pathologies (neural tube defects, colon cancer) as described in the proposal. We are in the progress of generating constructs that will be used to alter intracellular folate concentration as occurs during pregnancy and tumor progression. We have cloned the murine heavy chain ferritin gene as well as the murine 10-formyltetrahydrofolate dehydrogenase gene. In the coming year, we will generate mice with increased heavy chain ferritin expression, and mice with decreased 10-formyltetrahydrofolate dehydrogenase expression. Both of these new
mouse strains are expected to exhibit symptomatic folate deficiency independent of folate intake. The role of tissue specific folate deficiency on folate-associated related pathologies will be performed as described above.
Impacts
Impairments of folate metabolism are associated with many pathologies including cancer, birth defects and anemia. These studies will determine the role of critical genes that influence or mediate the effects of folate metabolism on these pathologies. From these studies, it is anticipated that new preventative strategies for folate-related disease will emerge.
Publications
- Liu, X., Szebenyi, D. M. E., Thiel, D. and Stover, P. J. 2001. Lack of catalytic Activity of a Murine mRNA Cytoplasmic Serine Hydroxymethyltransferase Splice Variant: Evidence against Alternative Spliceing as a Regulatory Mechanism Biochemistry 40, 4932-4939.
- Oppenheim, E., Nasrallah, I. M., Johnson, C., Mastri, M., and Stover, P. J. 2001. Heavy Chain Ferritin Enhances Cytoplasmic Serine Hydroxymethyltransferase Expression and de novo Thymidine Biosynthesis. J. Biol. Chem. 276, 19855-19861.
- Zhao, R., Chen, Y., Tan, W., Sharma, A., Stover, P., Rosowsky, A. and Deth, R. C. 2001. Relationship Between Dopamine-Stimulated Phospholipid Methylation and Single-Carbon Folate Pathway. J. Neurochem. 78, 1-10.
- Hirschberger, L. L., Daval, S., Stover, P. J. and Stipanuk, M. H. 2001. Murine Cysteine Dioxygenase Gene: Structural Organization, Tissue-specific Expression and Promoter Identification. Gene 277:153-61.
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Progress 01/01/00 to 12/31/00
Outputs
The studies outlined in this proposal seek to elucidate the role of key genes that regulate folate metabolism in folate-associated pathologies. Folate metabolism is required for the synthesis of DNA precursors, DNA methylation and numerous other cellular methylation reactions. Disruption of folate metabolism, either by folate deficiency or due to genetic polymorphisms, results in identifiable pathologies including anemias, neural tube defects and certain cancers. Our laboratory has identified several genes, including serine hydroxymethyltransferase, heavy chain ferritin and 10-formyktetrahydrofolate dehydrogenase, that regulate folate status and folate metabolism. Using internal ribosome entry sequences (IRES) and Cre/lox technologies, we have created a DNA construct that enables cell-specific and temporal deletion of the cytoplasmic serine hydroxymethyltransferase (cSHMT) gene. We have used this construct to generate murine embryonic stem cells (ES cells) that
contain an altered cSHMT gene such that the gene can be conditionally deleted in a whole animal. Using microinjection techniques, chimeric mice were generated from these modified ES cells and bred. We have screened 30 progeny and to date generated one germ line hetrozygote mouse that contains the modified cSHMT gene. We assume that these mice will have impaired homocysteine remethylation capacity, decreased DNA methylation, and exhibit pathologies associated with these biochemical markers. Once the colony is established, we will be able to rapidly delete the cSHMT gene selectively in any desired tissue and determine it's effects on folate-associated pathologies as described in the proposal. We are in the progress of generating constructs that will be used to alter intracellular folate concentration as occurs during pregnancy and tumor progression. We have cloned the murine heavy chain ferritin gene as well as the murine 10-formyltetrahydrofolate dehydrogenase gene. In the coming year,
we will generate mice with increased heavy chain ferritin expression, and mice with decreased 10-formyltetrahydrofolate dehydrogenase expression. Both of these new mouse strains are expected to exhibit symptomatic folate deficiency independent of folate intake. The role of tissue specific folate deficiency on folate-associated related pathologies will be performed as described above.
Impacts
Impairments of folate metabolism are associated with many pathologies including cancer, birth defects and anemia. These studies will determine the role of critical genes that influence or mediate the effects of folate metabolism on these pathologies. From these studies, it is anticipated that new preventative strategies for folate-related disease will emerge.
Publications
- Liu, X., Szebenyi, D. M. E., Thiel, D. and Stover, P. J. (2001) Cytoplasmic Serine Hydroxymethyltransferase: Expression, Structure and Function of an Alternative mRNA Splice Variant (in press, Biochemistry)
- Szebenyi, D. M. E., Liu, X., Kriksunov, I., Stover, P. J., and Thiel, D. (2000) X-ray Structure of the Murine Cytoplasmic Serine Hydroxymethyltransferase Quinonoid Ternay Complex: Evidence for Asymmetric Obligate Dimer Pairs. Biochemistry 39, 13313-13323.
- Suh, J. R., Oppenheim, E. W., Girgis, S. and Stover, P. J. (2000) Purification and Properties of a Folate Catabolizing Enzyme. J. Biol. Chem. 275, 35646-35655.
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