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December 1985, Volume 35, Issue 12

Editorial

MECHANISMS OF RESISTANCE TO METHOTREXATE

M. Perwaiz Iqbal  ( Department of Biochemistry, The Aga Khan University Medical College, Karachi. )

Methotrexate (MTX) is a folate antagonist which kills the proliferating cell by binding tightly to the enzyme dihydrofolate reductase (DHFR). Due to this binding the pathway of de novo DNA synthesis is blocked1-3. The drug has produced spectacular results controlling choriocarcinoma, Burkitt’s lymphoma, acute leukemia and-psoriasis. But continued administration to patients often results in emergence of drug-resistance. Because of this problem investigators have, over the past two decades, studied a variety of possible mechanisms for the development of resistance. As a result of these intensive studies, five mechanisms have been put forward.
The first mechanism involves a structural change in the enzyme, DHFR, such that the normal high affinity for MTX is lost. When this happens the enzyme would not be inhibited by the drug. Evidence for this mechanism has been reported by a number of laboratories4-9 . Albrecht and his associates by a multi-step selection procedure have isolated a methotrexate-resistant Chinese hamster cell line which contained structurally altered DHFR9. The mutant enzyme had a single pH optimum for the reduction of dthydrofolate as compared to double pH optima observed in the wild-type enzyme. The mutant enzyme was also found to have a dramatically altered affinity for MTX. It was shown that to achieve a 50% inhibition of the enzyme from the mutant cells, a 10-fold excess of MTX was required when compared to the amount of drug required to achieve a 50% inhibition of enzyme activity in parental cells.
In the second mechanism there is a structural change in a transport protein for MTX which is located in the surface membrane of the cell. In this case, the uptake of the drug by the cell may be remarkably reduced and the enzyme inside the cell again would not be inhibited. With regards to this mechanism of resistance, evidence has been provided by Sirotank and his associates who succeeded in isolating mutants of D. pneumoniae exhibiting levels of resistance to MTX of up to 100-fold as compared to the wild type strain10.
The uptake of the drug by these mutants was remarkably slow and far less than the wild type indicating that the resistance was due to changes in the transport system of the drug. Similar evidence has been presented by Hill and his associates11.
The third possible mechanism is an increase in the levels of DHFR in the cells. The sensitivity of the enzyme towards the drug remains the same. However there would be an excess of the enzyme with respect to the concentration of the drug in the cells, and some of the enzyme would be available to reduce dihydrofolate into tetrahydrofolate and hence, the pathway of de novo DNA synthesis would continue. Evidence for this mechanism of resistance has been reported by a number of laboratories12-24 Schimke, Bertino and their associates have shown that although the increased level of the enzyme, DHFR, can be due to decreased catabolism of the enzyme due to its stabilization as a result of binding to the inhibitor, yet enzyme induction is the major cause of this increase in the enzyme levels12 This enzyme induction takes place as a result of gene amplification, the process whereby a small part of the genome, representing one or more genes, is duplicated locally within a chromosome12,13 Such an amplification of genes is stable if it is localized in a specific region of a chromosome or unstable if localized in the nucleus as extrachromosomal DNA13,25.
The fourth possible mechanism of drug resistance is decreased polyglutamation of MTX inside the cell. Since polyglutamation facilitates longer retention of the drug by the cells, a decrease in that would result in rapid efflux of the drug and the enzyme, DHFR, would not be completely inhibited by the drug. Cowan and Jolivet have shown that the resistance to MTX exhibited by a human breast cancer cell line was due to decreased formation of MTX polyglutamates in these cells26.
The fifth possible mechanism of resistance to methotrexate is due to the induction of another molecular form of diliydrofolate reductase exhibiting low affinity for methotrexate. There is some evidence that methotrexate resistant murine leukemia cells have increased levels of a form of DHFR having low affinity for MTX27,28.
Overproduction of this low affinity form of enzyme has also been shown in cultured mouse fibroblasts and Chinese hamster cells29,30.
These studies for the elucidation of the mechanism of resistance to MIX are important because they give us a basis for a better understanding of general mechanisms of drug resistance. They also provide us with a rationale for the principles of cancer chemotherapy. For example, combination chemotherapy in most neoplastic diseases is usually considered to be superior to single drug treatments because in combination chemotherapy most of the neoplastic cells are likely to be killed and the chances for the development of resistance to the drugs are considerably reduced. It also tells us that a treatment should be carried out only as long as is necessary and the thug should be immediately changed if resistance develops. Over exposure of the cells with the drug is going to impart a stable form of resistance to the drug and, hence, must be avoided.

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