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Methotrexate resistance in L5178Y mouse leukemia cells Dedhar, Shoukat


Methotrexate, a folic acid antagonist, has been used in the clinical treatment of a wide variety of malignant neoplasms for over 20 years, either as a single agent or in combination with other antineoplastic agents. It is a cell cycle specific inhibitor and kills cells only in the S phase of growth. MTX is a potent inhibitor of the enzyme dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP⁺ oxidoreductase, EC, which catalyses the NADPH dependent reduction of dihydrofolic acid and folic acid to tetrahydrofolic acid: the metabolically active coenzyme form of folic acid essential in the biosynthesis of dTMP from dUMP by thymidylate synthetase. Inhibition of DHFR therefore leads to the inhibition of DNA synthesis and cell death. Methotrexate has many favourable properties; for instance, it interacts directly with intracellular sites without the need for prior metabolic transformation. It can be administered in large doses because toxicity to normal cells can be minimized by the administration of folinic acid (N5 formyl tetrahydrofolic acid) shortly after the administration of MTX. However, the effectiveness of MTX is inevitably compromised by the emergence of drug resistance, which can be either intrinsic, i.e. the tumour cells are resistant to MTX at the outset, or the tumour cells acquire resistance after exposure to MTX. An understanding of the mechanisms of resistance to MTX is therefore very important if treatment with this potent antineoplastic agent is to be improved. Three mechanisms of resistance to MTX have been determined from studies with experimental tumour systems: impaired uptake of MTX; increased levels of dihydrofolate reductase; and appearance of altered dihydrofolate reductase with a lower affinity for MTX. Impaired uptake of MTX and increased levels of OHFR can both theoretically be overcome by sustaining increased concentrations of free intracellular MTX. This can be achieved by exposing the cells to higher concentrations of MTX, and many chemotherapeutic regimens now use 'high-dose' MTX which can achieve plasma concentrations of MTX as high as 10⁻³M. However, resistance to MTX is still a major clinical problem and the use of 'high-dose' MTX has not significantly increased the therapeutic index of MTX treatment. Appearance of DHFR with a lower affinity for MTX suggests as an alternative the synthesis of an agent which would be a potent inhibitor of the altered enzyme, and requires the detailed characterization of the properties of this enzyme. If the altered enzyme retains some affinity for MTX, the administration of MTX and the more potent agent would result in better growth inhibition of the resistant tumour. In this thesis, a mouse leukemia cell line (L5178Y) grown in suspension culture was used to isolate two MTX-resistant cell lines and these were used to study the mechanisms leading to MTX resistance. Both resistant cell lines exhibited impaired MTX uptake when exposed to 10⁻⁶M MTX but not when exposed to 10⁻⁴M MTX, Both lines also had elevated DHFR levels (7 to 9 fold). A variant form of DHFR present in small amounts in both cell lines was isolated by MTX-sepharose affinity chromatography. The altered DHFR differed from the major form of reductase present in these cells in its markedly lower affinity (100,000 fold) for MTX. The two forms of the enzyme were purified from the most resistant cell line and their properties compared. They were found to differ moderately in their Km for substrates, however, the Ki of MTX differed by a factor of 100,000 for the two forms. In addition there were marked differences in their heat stability, isoelectric points and sensitivity to p-chloromercuriphenyl-sulphonate, and a minor difference in their molecular weights. It is concluded that the presence of a highly resistant form of DHFR in these cell lines represents an important mechanism in conferring a high degree of resistance to these cells. The importance of this form of DHFR in MTX resistance is discussed in relation to impaired transport and elevated DHFR levels. Experiments to determine the amino acid sequence of the altered enzyme are underway and once determined should facilitate the synthesis of specific inhibitors of its activity.

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