Mutations in the gene for human dihydrofolate reductase: an unlikely cause of clinical relapse in pediatric leukemia after therapy with methotrexate.
Resistance to methotrexate (MTX) in some sublines of mammalian cells is reported to be due to one of the following amino acid substitutions in dihydrofolate reductase (DHFR) that lower inhibition by MTX: Gly15 to Trp, Leu22 to Arg or Phe or Phe31 to Trp or Ser. We have produced variants of human DHFR (hDHFR) with these substitutions by directed mutagenesis. Recombinant hDHFR variants expressed in Escherichia coli have greatly decreased inhibition by MTX, but decreased catalytic efficiency, and in one case decreased stability. When a retroviral vector encoding wild-type (wt) hDHFR or one of these variants was introduced into murine fibroblasts or bone marrow progenitors, modest protection from MTX was conferred, even by wt. Relapsed pediatric patients with acute lymphoblastic leukemia who have received multiple courses of high-dose MTX seem most likely to develop such MTX resistance. cDNA was reverse transcribed from blast mRNA from 17 of these patients. However, upon amplification and sequencing of DHFR cDNA, no resistance mutation was found. The explanation for this probably lies in the need for considerable gene amplification to offset lowered catalytic efficiency, and the need for two-base changes for most substitutions, both of which are probably infrequent events.
Spencer
H T
HT
Department of Molecular Pharmacology, St Jude Children's Research Hospital, Memphis, Tennessee, USA.
Sorrentino
B P
BP
Pui
C H
CH
Chunduru
S K
SK
Sleep
S E
SE
Blakley
R L
RL
D000293:Adolescent; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000627:therapeutic use*; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D019008:Drug Resistance, Neoplasm; D005260:Female; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D007223:Infant; D008297:Male; D008727:Methotrexate / Q000627:therapeutic use*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009154:Mutation; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics*; D012008:Recurrence; D012190:Retroviridae / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification
D000964:Antimetabolites, Antineoplastic; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate