Methotrexate-resistant Chinese hamster ovary cells contain a dihydrofolate reductase with an altered affinity for methotrexate

Abstract

Previous reports [Flintoff, W. F., Davidson, S. V., & Siminovitch, L. (1976) Somatic Cell Genet. 2, 245--261; Gupta, R. S., Flintoff, W. F., & Siminovitch, L. (1977) Can. J. Biochem. 55, 445--452] described a series of Chinese hamster ovary cells that were resistant to the cytotoxic action of methotrexate and contained a dihydrofolate reductase that was less sensitive to inhibition by the drug than wild-type enzyme. In this study, binding of labeled methotrexate to the reductase--NADPH complex and separation of free and bound drug by filtration through Sephadex G--25 have been used to demonstrate that clonal isolates of these resistant cells contain a dihydrofolate reductase varying between 2.5- and 6-fold lower in affinity for the drug than the wild-type enzyme. The apparent dissociation constant for the wild-type enzyme is 0.5 x 10(-9) M. Using two-dimensional polyacrylamide gel electrophoresis, 11 independently selected resistant isolates have been shown to contain a reductase with a similar overall net charge as the wild-type enzyme. Reductase purified from either wild-type or resistant cells contains two components after isoelectric focusing in polyacrylamide gels. The major component represents about 90% of the total protein and has a pI of about 8.0. The minor component representing about 10% of the reductase protein has a pI between 7.2 and 7.6.

A study of monoclonal antibody‐producing CHO cell lines: What makes a stable high producer?

“…However, the specific productivity decreased upon further increase of MTX level (Kim et al, 2001). Gene amplification by MTX selection is generally obtained by gradual increase of MTX selection pressure rather than rapid increase as single-step high-level resistance to MTX may result in mechanisms other than dhfr-mediated gene amplification, such as altered MTX transport properties (Assaraf and Schimke, 1987;Kaufman, 1990;Sirotnak et al, 1981) or an altered, MTX-resistant DHFR enzyme (Flintoff and Essani, 1980;Flintoff et al, 1976;Haber and Schimke, 1981). Hence, a gradual increase, instead of a rapid increase, of MTX concentration is more effective for constructing high-producer recombinant CHO cell lines as previously shown by Nakanishi et al (1997) and Yoshikawa et al (2000a).…”

The dihydrofolate reductase amplicons in different methotrexate-resistant Chinese hamster cell lines share at least a 273-kilobase core sequence, but the amplicons in some cell lines are much larger and are remarkably uniform in structure.

“…1 and 2A) (21,22 [32]) and contain 5 to 60 copies of the DHFR gene per diploid nucleus (9,11,12). The RI cell line and its derivatives, RIII-1 and RIII-2, contain an altered DHFR gene that encodes an enzyme with a lowered affinity for MTX (10,17 variant fragments that contain junctions would have been observed (i.e., if a sequence is duplicated and then joined end-to-end, two parental-sized restriction fragments will be united at the joint to form a new restriction fragment of a different size). We conclude that the major amplicon types in the RIII-1 and RIII-2 cell lines are at least as large as the 273-kb type I amplicon cloned from CHOC 400 and represent nonrearranged versions of the parental sequence.…”

Moderate-level gene amplification in methotrexate-resistant Chinese hamster ovary cells is accompanied by chromosomal translocations at or near the site of the amplified DHFR gene.

“…A class of Mtx-resistant Chinese hamster ovary (CHO) cells with a moderate level of amplification has been described previously (17). These lines, termed class III, were derived from class I mutants that were already somewhat resistant to Mtx by virtue of an alteration in the dhfr structural gene rendering it more resistant to the drug (14,15). The RIII-type mutants displayed a moderate level of amplification of the abnormal dhfr gene (17), resulting in moderate overproduction of the resistant enzyme (14,17).…”