Abstract
The Chinese hamster lung cell line DC-3F contains two polymorphic dihydrofolate reductase (DHFR) alleles that are defined by an Asp-Asn amino acid sequence difference at position 95 in the protein. Previously, we reported that the antifolate-resistant subline DC-3F/A3 overexpressed a Leu22 → Phe mutant of the Asp95 (21k) allele and that this was the basis of its resistance to methotrexate (MTX) and methasquin [P. W. Melera, J. P. Davide, C. A. Hession, and K. W. Scotto. Mol. Cell. Biol., 4: 38–48, 1984]. We now show that another independently selected antifolate-resistant subline of DC-3F, DC-3F8/A55, in addition to being severely compromised in its ability to accumulate MTX, overexpresses a Leu22 → Phe mutant form of the Asn95 (20k) allele. Characterization of purified DHFR from these cells showed that the enzyme displayed a 6-fold higher Kd for MTX (3.92 ± 0.17 pm) than the wild type (0.58 ± 0.10 pm), thus explaining its lowered sensitivity to drug. Unexpectedly, however, this value was 4-fold lower than that displayed by the DC-3F/A3 enzyme even though both contain the same (Leu22 → Phe) mutation and differ only at position 95. Indeed, we have also shown that the 21k and 20k wild type enzymes, both containing Leu at position 22, in fact differ by 3-fold (1.58 ± 0.08 and 0.58 ± 0.10 pm, respectively) in their Kd's for MTX. This demonstrates that the amino acid at position 95 has an effect on the ability of DHFR to bind MTX. On the other hand, these allelic variants are indistinguishable from each other in their catalytic properties and in their respective Kd's for dihydrofolate. Taken together, these characteristics are consistent with the observation that it is the wild type 21k allele which is preferentially overexpressed at a frequency of 3:1 in MTX-resistant Chinese hamster lung sublines derived by long-term selection in MTX.
The results of these studies are novel in that they establish a role for allelic variation in the DHFR gene as a contributor to antifolate resistance in mammalian cells. Moreover, they implicate amino acid position 95 in the maintenance of the structure of the MTX binding pocket.
Footnotes
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↵1 This work was supported in part by NIH Grant 49538 to P. W. M. and by SAPEC S.A. Fine Chemicals.
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↵2 To whom requests for reprints should be addressed, at 108 N. Greene Street, Graduate Program in Molecular and Cell Biology, University of Maryland, Baltimore, MD 21201.
- Received July 28, 1993.
- Accepted October 12, 1993.
- ©1993 American Association for Cancer Research.
Volume 53, Issue 24
