Abstract
To elucidate molecular mechanism(s) of cellular response to mercaptopurine, a widely used antileukemic agent, we assessed mercaptopurine (MP) sensitivity in mismatch repair (MMR) proficient and MMR deficient human acute lymphoblastic leukemia (ALL) cells. Sensitivity to thiopurine cytotoxicity was not dependent on MMR (i.e., MutSα) competence among six cell lines tested. Using electrophoretic mobility shift assay analysis, we found that the incubation of nuclear extracts from ALL cells with synthetic 34-mer DNA duplexes containing deoxythioguanosine (GS) within either GS·T or GS·C pairs, resulted in formation of a DNA-protein complex distinct from the DNA-MutSα complex and unaffected by ATP. Isolation and sequence analysis of proteins involved in this DNA-protein complex identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a component. Western blot analysis of nuclear extracts from a panel of human lymphoblastic leukemia cell lines revealed markedly different basal levels of GAPDH in nuclei, which was significantly related to thiopurine sensitivity (p = 0.001). Confocal analysis revealed markedly different intracellular distribution of GAPDH between nucleus and cytosol in six human ALL cell lines. Redistribution of GAPDH from cytosol to nucleus was evident after MP treatment. These findings indicate that a new DNA-protein complex containing GAPDH and distinct from known MMR protein-DNA complexes binds directly to thioguanylated DNA, suggesting that this may act as a sensor of structural alterations in DNA and serve as an interface between these DNA modifications and apoptosis.
Footnotes
- Received June 2, 2000.
- Accepted October 6, 2000.
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Send reprint requests to: Dr. William E. Evans, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38163. E-mail: william.evans{at}stjude.org
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This study was supported by National Institutes of Health Grants R37-CA36401, R25–23944, and Cancer Center Support Grant CA21765; by a Center of Excellence Grant from the State of Tennessee; and by the American Lebanese Syrian Associated Charities (ALSAC).
- The American Society for Pharmacology and Experimental Therapeutics
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