Identification of single nucleotide polymorphisms in the human gamma-glutamyl hydrolase gene and characterization of promoter polymorphisms.
gamma-Glutamyl hydrolase (GGH) plays a central role in folate metabolism and antifolate action. Increased GGH activity has been found in rat hepatoma cells resistant to the cancer drug methotrexate (MTX). The aim of this study was to identify polymorphisms in the GGH gene that modulate GGH activity and that may affect methotrexate resistance. Exons of the human gamma-glutamyl hydrolase (hGGH) gene were amplified by polymerase chain reaction (PCR) from breast cancer tissue and leukemia cell lines. Single-stranded conformational polymorphism (SSCP) analysis was performed, and PCR products containing different patterns were cloned and sequenced. Six single nucleotide polymorphisms (SNPs) were identified, at bases -401C>T, -354G>T, -124T>G, +16T>C, +452C>T, and +1102A>G, relative to the A of the translation start codon being considered as +1. The SNP at +16, which changes codon -19 (relative to the start of the mature hGGH protein) in the endoplasmic reticulum targeting sequence of hGGH protein from cysteine to arginine, has previously been identified in this laboratory. The SNP at +452 changes the conserved hGGH protein codon 127 from threonine to isoleucine. The functions of SNPs in the promoter of the hGGH gene were studied by site-directed mutagenesis of a 516-bp region of the hGGH gene promoter in a luciferase reporter vector and transfection into HepG2 and MCF-7 cells. All of the promoter polymorphisms enhanced the production of luciferase compared to the wild-type hGGH gene promoter in HepG2 cells, and -401C>T and -124T>G enhanced luciferase expression in MCF-7 cells, suggesting that polymorphisms in the hGGH gene promoter may increase expression of hGGH protein.
Chave
Karen J
KJ
Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, PO Box 509, Albany, NY 12201-0509, USA. chave@wadsworth.org
Ryan
Thomas J
TJ
Chmura
Stacey E
SE
Galivan
John
J
D002460:Cell Line; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018922:HL-60 Cells; D006801:Humans; D019169:Jurkat Cells; D020014:K562 Cells; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D011623:gamma-Glutamyl Hydrolase / Q000235:genetics* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D004247:DNA; D008156:Luciferases; D011623:gamma-Glutamyl Hydrolase