Molecular basis for decreased folylpoly-gamma-glutamate synthetase expression in a methotrexate resistant CCRF-CEM mutant cell line.
A CCRF-CEM mutant, CEM-p, has been shown to exhibit resistance to methotrexate due to decreased methotrexate polyglutamate accumulation. To ascertain the mechanism(s) responsible for this phenotype, we analyzed FPGS and SLC19A1 mRNA expression, examined FPGS promoter activity, and determined nucleotide sequence of the FPGS promoter and full length cDNA from CCRF-CEM and CEM-p cells. We identified in FPGS from CEM-p cells three amino acid substitutions that altered the ATP binding P-loop, glutamate/folate binding, and a conserved domain located at the carboxyl-terminal. Our data demonstrated for the first time the importance of the highly conserved domain (VTGSLHLVGGV) located at the carboxyl-terminal for FPGS activity.
Leclerc
Guy J
GJ
Department of Pediatrics, Medical University of South Carolina, PO Box 250514, Charleston, SC 29425, USA.leclergj@musc.edu
York
Teresa A
TA
Hsieh-Kinser
Tingting
T
Barredo
Julio C
JC
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D004789:Enzyme Activation / Q000187:drug effects; D020869:Gene Expression Profiling; D006801:Humans; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000201:enzymology* / Q000235:genetics* / Q000628:therapy; D026901:Membrane Transport Proteins / Q000235:genetics; D008727:Methotrexate / Q000494:pharmacology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D010453:Peptide Synthases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics; D058978:Reduced Folate Carrier Protein; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013329:Structure-Activity Relationship
D026901:Membrane Transport Proteins; D012333:RNA, Messenger; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D008727:Methotrexate