Methotrexate resistance in an in vivo mouse tumor due to a non-active-site dihydrofolate reductase mutation Journal Article

   


Authors: Dicker, A. P.; Waltham, M. C.; Volkenandt, M.; Schweitzer, B. I.; Otter, G. M.; Schmid, F. A.; Sirotnak, F. M.; Bertino, J. R.
Article Title: Methotrexate resistance in an in vivo mouse tumor due to a non-active-site dihydrofolate reductase mutation
Abstract: A series of methotrexate (MTX)-resistant L1210 leukemia murine ascites tumors were developed in vivo and analyzed for drug resistance. Three of 20 tumors studied expressed an altered dihydrofolate reductase (DHFR) and each was identical, having a C to T base transition at nucleotide 46 in the DHFR gene as demonstrated by PCR and direct sequencing. This transition results in a Gly to Trp substitution at amino acid 15 of the enzyme. Purified altered enzyme displays significantly lower binding affinity for the antifolates MTX, trimetrexate, edatrexate, and trimethoprim with respective Ki values 165-, 76-, 30-, and 28-fold higher than values obtained for enzyme isolated from parental tumor (wild-type enzyme). Substrate (dihydrofolate) and cofactor (NADPH) binding is also diminished for the mutant enzyme, although to a lesser extent (17.3- and 3.6-fold higher Km, respectively). Gly-15 is highly conserved for all vertebrate species of DHFR but has no known interaction(s), either directly or indirectly, with bound cofactor, substrate, or inhibitor. Protein molecular modeling reveals that the affected residue is 9-12 Å away from the enzyme active site and located in a region analogous to the mobile Met-20 loop domain characterized for Escherichia coli DHFR.
Keywords: leukemia; gene mutation; nonhuman; methotrexate; polymerase chain reaction; protein conformation; animal cell; mouse; animal; mice; animal model; drug resistance; time factors; animalia; vertebrata; amino acid sequence; molecular sequence data; kinetics; escherichia coli; dna, neoplasm; murinae; base sequence; models, molecular; binding sites; dna primers; point mutation; dihydrofolate reductase; tetrahydrofolate dehydrogenase; enzyme substrate complex; ascites tumor; enzyme active site; nadp; michaelis constant; enzyme stability; trimethoprim; nucleic acid base substitution; trimetrexate; chromatography, affinity; reduced nicotinamide adenine dinucleotide phosphate; leukemia l1210; edatrexate; human; priority journal; article
Journal Title: Proceedings of the National Academy of Sciences of the United States of America
Volume: 90
Issue: 24
ISSN: 0027-8424
Publisher: National Academy of Sciences  
Date Published: 1993-12-15
Start Page: 11797
End Page: 11801
Language: English
DOI: 10.1073/pnas.90.24.11797
PUBMED: 8265628
PROVIDER: scopus
PMCID: PMC48071
DOI/URL:

https://www.scopus.com/inward/record.uri?eid=2-s2.0-0027142053&doi=10.1073%2fpnas.90.24.11797&partnerID=40&md5=fdbbbbca147ca296494ec75fee0a841f
Notes: Article -- Export Date: 1 March 2019 -- Source: Scopus
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MSK Authors
  1. Joseph Bertino
    363 Bertino
  2. Francis M Sirotnak
    184 Sirotnak
  3. Glenys M. Otter
    27 Otter
  4. Mark C. Waltham
    23 Waltham
  5. Matthias Volkenandt
    27 Volkenandt
  6. Adam P. Dicker
    15 Dicker
  7. Barry I. Schweitzer
    22 Schweitzer
  8. Franz A. Schmid
    26 Schmid
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