Pharmacological and molecular investigations on the mechanisms underlying resistance of human leukaemia cells to the antimetabolites methotrexate, 6-mercaptopurine and 6-thioguanine

Abstract

The goal of this thesis has been to elucidate the mechanisms underlying resistance to methotrexate (MTX), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), the antimetabolites widely used in treatment of childhood leukemia, as well as to determine the influence of 7-hydroxymethotrexate (7-OHMTX), the major metabolite of MTX, on the therapeutic action of MTX.

Resistant sublines of leukemic cell lines were developed by long-term exposure to stepwise increasing concentrations of these different agents. The mechanism underlying resistance to MTX in cells exposed to this drug was a pronounced reduction (> 10-fold) in reduced folate carrier (RFC) mediated uptake of MTX. In CCRF-CEM cells, this reduction was associated with transcriptional silencing of the RFC gene, due to attenuated or even abolished binding of various transcription factors to their cis-acting elements, including the CRE, E-box, AP1, Mzf-1 and GC-box. In contrast, resistance to 7-OHMTX was due solely to a dramatic decrement (> 95%) in folylpolyglutamate synthetase activity, which also conferred a greater than 100-fold increase in resistance to short-term exposure to MTX.

The levels of mRNA species originating from approximately 17000 genes present in MTX- and 7-OHMTX-resistant MOLT4 cells were compared. In the MTX-resistant subline, the levels of mRNA encoding proteins involved in DNA and RNA metabolism and in transport were altered most; whereas in the 7-OHMTX-resistant cells, mRNA species associated with metabolism and cell proliferation were affected more profoundly. In these 7-OHMTX-resistant cells, the 10-fold reduction in the level of the mRNA for adenosine deaminase (the major enzyme of purine catabolism), complete absence of mRNA for cystathionine â synthase, the 3-fold higher level of the mRNA for methylene tetrahydrofolate reductase (involved in methyl-tetrahydrofolate biosynthesis) and the 2-fold elevation in the level of the mRNA for glycinamide ribonucleotide formyltransferase (involved in purine biosyntheses), all revealed a pattern of preservation of pools of intracellular folates and of nucleotide biosynthesis.

Neither of the known mechanisms of resistance to thiopurines (i.e., alterations in the activity of hypoxanthine guanine phosphoribosyl transferase or of thiopurine methyltransferase enzymes) was found to occur in 6-MP- or 6-TG-resistant cells. Instead, the primary mechanism of resistance was a pronounced reduction in cellular uptake of 6-MP. Selective down-regulation of the levels of mRNAs encoding two nucleoside transporters, the concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2), was detected in both resistant sublines. Moreover, silencing of the CNT3 and ENT2 genes by small interfering RNA attenuated both the transport and cytocidal effect of 6-MP.

Both of the thiopurine-resistant cell sublines exhibited a collateral enhancement in sensitivity to the cytotoxicity of methylmercaptopurine riboside (meMPR), an intra-cellular metabolite of 6-MP that is known to be a potent inhibitor of de novo purine biosynthesis. Transport of meMPR into these cells remained intact. These findings, together with the reduced rate of de novo purine biosynthesis and low levels of ribonucleoside triphosphates in these cells, can easily explain their enhanced sensitivity to meMPR. An additional investigation revealed that transfection of wild-type cells with small interference RNA molecules targeting the gene encoding the first member of the family of equilibrative nucleoside transporters (ENT1) reduced the initial rate of meMPR uptake.

In summary, our present findings clearly demonstrate the major involvement of defective transport in the development of resistance to MTX, 6-MP and 6-TG. Long-term exposure of leukemic cells to 7-OHMTX can impair the clinical efficacy of MTX. The disparate patterns of gene expression exhibited by MTX- and 7-OHMTX-resistant cells further confirms that these agents act in different ways. These results may help to improve individualization of MTX treatment on the basis of plasma levels of 7-OHMTX. The collateral enhancement in the sensitivity of thiopurine-resistant cells to the cytotoxicity of meMPR suggests that administration of meMPR or its analogues to patients with ALL experiencing relapse or resistance might be beneficial.