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In vitro treatment of human T-cell leukemia cells with 7-hydroxymethotrexate, the major metabolite of methotrexate resulted in acquired resistance as a result of the complete loss of folypolyglutamate synthetase (FPGS) activity. This was in contradistinction to the major modality of antifolate resistance of impaired drug transport in leukemia cells exposed to methotrexate. To identify the genes associated with methotrexate and 7-hydroxymethotrexate resistance, we herein explored the patterns of genome-wide expression profiles in these antifolte-resistant leukemia sublines. mRNA levels of the reduced folate carrier, the primary influx transporter of folates and antifolates, were down-regulated more than two-fold in methotrexate-resistant cells. The dramatic loss of FPGS activity in 7-hydroxymethotrexate-resistant cells was associated with alterations in the expression of various genes aimed at preserving reduced folates and/or enhancing purine nucleotide biosynthesis, e.g. methylene tetrahydrofolate reductase, glycinamide ribonucleotide formyltransferase, adenosine deaminase, cystathionine β synthase, as well as the ATP-dependent folate exporters BCRP/ABCG2 and MRP1/ABCC1. The observed changes in gene expression were generally not paralleled by acquired DNA copy numbers alterations, suggesting transcriptional regulatory mechanisms. Interestingly, gene expression of DNA/RNA metabolism and transport genes were more profoundly altered in methotrexate-resistant subline, whereas in 7-hydroxymethotrexate-resistant cells, the most profoundly affected groups of genes were those encoding for proteins involved in metabolism and cellular proliferation. Thus, the present investigation provides evidence that 7-hydroxymethotrexate induces gene expression alterations and an antifolate resistance modality that are distinct from its parent drug methotrexate.