Defense Date

2008

Document Type

Thesis

Degree Name

Master of Science

Department

Pharmacology & Toxicology

First Advisor

Rechard G Moran

Abstract

Abstract: Folylpoly-γ-glutamate synthetase (FPGS) catalyzes the formation of polyglutamate forms of the reduced folates and antifolates such as methotrexate (MTX) and pemetrexed; this allows the retention of folates and antifolate cancer drugs inside the cell. The enzyme activity of FPGS is essential for cell proliferation and survival. The mouse fpgs gene contains two promoters spaced 10 kb apart which are activated in a tissue-specific manner. The upstream promoter (P1) and exons A1a and A1b are used in some differentiated tissues, mainly liver and kidney, whereas the downstream promoter (P2) and exon 1 are used in rapidly dividing cells. In contrast, the human fpgs gene expresses virtually all transcripts from the downstream promoter. In order to more faithfully mimic human folate metabolism in the mouse, we have deleted the upstream promoter and the associated two small exons of fpgs in the mouse genome by homologous recombination. Homozygous deletion mice survive embryonic development, grow to adulthood, and reproduce through several generations, they appear to be normal. The results of Q-RT-PCR analysis on RNA from adult mouse liver of three different genotypes (A1aA1b +/+, +/-, -/-) indicated that deletion of P1 results in the release of promoter interference of P2, and activation of the downstream P2 promoter is increased by 3-5 fold. Interestingly, the total FPGS mRNA expression in KO mouse liver is 20-100 fold lower than in liver from wild-type mice. However when the FPGS activity was measured using an FPGS enzyme assay, the liver of knockout mice appeared to have only 2 fold lower enzyme activity than liver from wild-type mice. In conclusion, we have successfully generated a mouse which reflects human folate metabolism much closer than seen in wild type mice. The FPGS- humanized mouse liver model would be an appropriate in vivo tool for the study of the antifolate drug toxicity and inhibition.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

December 2008

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