Abstract
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that acts at the intersection of energy metabolism and stress response in tumor cells. To elucidate the role of GAPDH in chemotherapy-induced stress, we analyzed its activity, protein level, intracellular distribution, and intranuclear mobility in human carcinoma cells A549 and UO31 after treatment with cytarabine, doxorubicin, and mercaptopurine. After treatment with cytosine arabinoside (araC), enzymatically inactive GAPDH accumulated in the nucleus. Experiments on fluorescence recovery after photobleaching with green fluorescent protein-GAPDH fusion protein in the live cells treated with araC demonstrated reduced mobility of green fluorescent protein-GAPDH inside the nucleus, indicative of interactions with nuclear macromolecular components after genotoxic stress. Depletion of GAPDH with RNA interference stopped cell proliferation, and induced cell cycle arrest in G1 phase via p53 stabilization, and accumulation of p53-inducible CDK inhibitor p21. Neither p21 accumulation nor cell cycle arrest was detected in GAPDH-depleted p53-null NCI-H358 cells. GAPDH-depleted A549 cells were 50-fold more resistant to treatment with cytarabine (1.68 ± 0.182 μM versus 0.03 ± 0.015 μM in control). Depletion of GAPDH did not significantly alter cellular sensitivity to doxorubicin (0.05 ± 0.023 μM versus 0.035 ± 0.0154 μM in control). Induction of cell cycle arrest in p53-proficient carcinoma cells via GAPDH abrogation suggests that GAPDH-depleting agents may have a cytostatic effect in cancer cells. Our results define GAPDH as an important determinant of cellular sensitivity to antimetabolite chemotherapy because of its regulatory functions.
- GAPDH, glyceraldehyde 3-phosphate dehydrogenase
- FRAP, fluorescence recovery after photobleaching
- NO, nitric oxide
- araC, cytosine arabinoside, 4-amino-1-[(2R,3S,4R,5R)-3,4-dihydroxy-5- (hydroxymethyl)oxolan-2-yl] pyrimidin-2-one;
- MP, 6-mercaptopurine, 3,7-dihydropurine-6-thione
- DOX, doxorubicin, (8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-l-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5, 12-naphthacenedione
- MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- siRNA, short interfering RNA
- siGAPDH, siRNAs targeted against GAPDH mRNA
- CDK, cyclin-dependent kinase
- DSB, double-strand break
- GFP, green fluorescent protein
- EGFP, enhanced green fluorescent protein
- RNAi, RNA interference
- Ab, antibody
- PCA, personal cell analyzer.
Footnotes
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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This work was supported in part by the National Cancer Institute [Grant R01-CA104729]; by Jayne Haines Center for Pharmacogenomics and Drug Safety of Temple University School of Pharmacy; and by Temple University Summer Research Award (to E.K.).
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The results of this study were presented in part at: Phadke M, Krynetskaia N, Krynetskiy E (2008) Glycolytic and nonglycolytic functions of GAPDH in cellular response to genotoxic drugs (Abstract), in Proceedings of the 99th Annual Meeting of the American Association for Cancer Research; 2008 April 12–16; San Diego, CA. Abstr 3341, American Association for Cancer Research, Philadelphia, PA. Phadke MS, Barrero C, Mishra A, Krynetskaia N, Merali S, Krynetskiy E (2009) Nuclear glyceraldehyde 3-phosphate dehydrogenase: a signaling molecule for cellular response to various anticancer drug therapies (Abstract), in Proceedings of the 100th Annual Meeting of the American Association for Cancer Research; 2009 April 18–22; Denver, CO. Abstr 3773, American Association for Cancer Research, Philadelphia, PA.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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ABBREVIATIONS:
- Received April 29, 2009.
- Accepted July 21, 2009.
- © 2009 by the American Society for Pharmacology and Experimental Therapeutics
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