Sensitization of human erythroleukemia K562 cells resistant to methotrexate by inhibiting IMPDH.
BACKGROUND
Methotrexate (MTX) is an inhibitor of dihydrofolate reductase (DHFR), an enzyme involved in nucleotide synthesis, and is widely used in therapy, but its efficacy is often compromised by the development of resistance in cancer cells. To identify potential targets as modulators in MTX chemotherapy, we determined gene expression profiles upon MTX treatment.


METHODS
Expression profiles in human erythroleukemia K562 cells were determined after short treatment with MTX or once resistance to MTX was established. Screening was performed using the Atlas Human Cancer 1.2K arrays (BD-Clontech) containing 1176 cancer-related cDNAs. Specific software (Atlas Image 2.0.1 and Gene Spring 6.1) was used to analyze the expression level for each gene in each condition. Results were validated by quantitative RT-PCR, and the cytotoxicity of the different treatments was assessed by the MTT assay.


RESULTS
Differentially expressed genes were selected considering a factor of 2 with respect to their expression in control cells. Eight genes were found to be overexpressed and 14 underexpressed both in MTX-treated and resistant cells. Among the overexpressed genes we chose 5'-monophosphate dehydrogenase (IMPDH2) for further validation. Changes in IMPDH2 mRNA levels were confirmed and functional analyses were performed by inhibiting IMPDH with either benzamide riboside, mycophenolic acid, or tiazofurin. These inhibitors sensitized resistant K562 cells to MTX cytotoxicity.


CONCLUSIONS
Expression of IMPDH2 is increased in K562 cells upon short treatment with MTX and once resistance to MTX is established. Benzamide riboside, mycophenolic acid, and tiazofurin could act as modulators of MTX chemotherapy in K562 cells.
Peñuelas
Silvia
S
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Barcelona, Spain.
Noé
Véronique
V
Morales
Raúl
R
Ciudad
Carlos J
CJ
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D020869:Gene Expression Profiling; D006801:Humans; D007168:IMP Dehydrogenase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020014:K562 Cells; D004915:Leukemia, Erythroblastic, Acute; D008727:Methotrexate / Q000494:pharmacology*; D020411:Oligonucleotide Array Sequence Analysis; D014158:Transcription, Genetic
D000964:Antimetabolites, Antineoplastic; D007168:IMP Dehydrogenase; C501687:IMPDH2 protein, human; D008727:Methotrexate