Resistance to methotrexate due to gene amplification in a patient with acute leukemia.

Abstract

A patient is described with acute myelocytic leukemia refractory to conventional therapy, who also became highly resistant to methotrexate (MTX) after repeated courses of this drug. Leukemia cells from this patient were found to contain an elevated level of dihydrofolate reductase (DHFR) activity, with no change in the affinity of the enzyme for MTX. A sensitive "dot blot" assay revealed a fourfold increase in the gene copy number of DHFR. Southern blot analysis with a human DHFR cDNA probe confirmed this increase in the gene copy number, and demonstrated a similar restriction pattern with Eco R1, Hind III, and Pst 1 as seen with a highly amplified human leukemia cell line, K562. Additional DHFR fragments were detected, not seen in the K562 blot, suggesting the presence of pseudogenes, or a result of gene rearrangements occurring as part of the amplification process. Resistance to MTX in this patient was therefore ascribed to gene amplification and overproduction of DHFR.

Digital karyotyping identifies thymidylate synthase amplification as a mechanism of resistance to 5-fluorouracil in metastatic colorectal cancer patients

“…For example, expression of multidrug resistance genes can confer resistance to drugs in vitro (31,32), but the relationship of such expression to the development of resistance in vivo remains conjectural. Similarly, the dihydrofolate reductase gene is commonly found to be amplified after treatment of cultured cells with methotrexate (33), but there are only a few case reports of gene amplification occurring in the tumors of cancer patients after exposure to conventional chemotherapeutic agents in vivo (22,34,35).…”

DHFR/MSH3 amplification in methotrexate-resistant cells alters the hMutSα/hMutSβ ratio and reduces the efficiency of base–base mismatch repair

“…However, human tumor cells can acquire resistance to Mtx, a phenomenon that has been demonstrated both in vitro and in vivo (5)(6)(7)(8). One well documented mechanism of resistance involves the amplification of a region of the human or rodent genome containing the DHFR gene (5)(6)(7)(8)(9)(10), an event that leads to elevated expression of DHFR, which effectively circumvents the metabolic block produced by this agent.…”

Identification of phosphorylase kinase as a novel therapeutic target through high-throughput screening for anti-angiogenesis compounds in zebrafish

“…Rescue of the phenotype observed under subsaturating concentrations of either compound by overexpression PhKG1 (through injection of mRNA) confirms that a component of the anti-angiogeneic effect of both compounds is dependent on inhibition of PhKG1. This rescue is analogous to drug resistance conferred by gene copy number amplification, such as clinical resistance to STI-571 due to amplifications in bcr-abl gene copy number (Gorre et al, 2001) and resistance to methotrexate in acute leukemia due to dihydrofolate reductase amplification (Carman et al, 1984;Goker et al, 1995) among others (Ferguson, 1991). The level of rescue obtained in the presence of F10 was substantially lower than that observed in the presence of F11, which is likely to reflect the stronger inhibitory effect of compound F11 on PhKG1 and the more pleiotropic nature of compound F10 (from the profiling data).…”