6-Thioguanine Reactivates Epigenetically Silenced Genes in Acute Lymphoblastic Leukemia Cells by Facilitating Proteasome-mediated Degradation of DNMT1
ABSTRACT Thiopurines including 6-thioguanine ((S)G), 6-mercaptopurine, and azathioprine are effective anticancer agents with remarkable success in clinical practice, especially in effective treatment of acute lymphoblastic leukemia (ALL). (S)G is understood to act as a DNA hypomethylating agent in ALL cells, however, the underlying mechanism leading to global cytosine demethylation remains unclear. Here we report that (S)G treatment results in reactivation of epigenetically silenced genes in T leukemia cells. Bisulfite genomic sequencing revealed that (S)G treatment universally elicited demethylation in the promoters and/or first exons of the genes that were reactivated. (S)G treatment also attenuated the expression of histone lysine-specific demethylase 1 (LSD1), thereby stimulating lysine methylation of the DNA methylase DNMT1 and triggering its degradation via the ubiquitin-proteasomal pathway. Taken together, our findings reveal a previously uncharacterized but vital mechanistic link between (S)G treatment and DNA hypomethylation.
SourceAvailable from: Senthil R Kumar[Show abstract] [Hide abstract]
ABSTRACT: Background The antimetabolite 6-thioguanine (6-TG) has been used to treat both human and canine lymphoid malignancies. 6-TG has been shown to be epigenetically active as a demethylating agent in a human lymphoma cell line, causing downregulation of DNA methyltransferase 1 (DNMT1) through ubiquitin-targeted degradation. Zebularine (Zeb), a similar cytidine analog, also has demethylating activity as well as oral bioavailability. The hypothesis of the present study was that 6-TG and Zeb would cause downregulation of DNMT1 and globally demethylate the genomic DNA of canine lymphoma cells. The secondary hypothesis was that these agents would cause a dose-dependent decrease in cell proliferation in canine lymphoma cells. Canine CLGL-90 malignant T cells and CLL 17¿7 cells were incubated in modified RPMI media. They were treated with 6-TG, Zeb, or control media at biologically relevant concentrations.ResultsFollowing treatment with each agent, DNMT1 protein and global DNA methylation were significantly decreased. A dose-dependent decrease in cell survival was also observed, with apoptosis being the primary mode of cell death in the CLGL-90 cell line.Conclusions These results confirm the demethylating action of 6-TG and Zeb in canine cells which is similar to that shown in human cell lines. Confirmation of this mechanism supports the clinical application of these compounds as demethylating drugs in veterinary patients.BMC Veterinary Research 12/2014; 10(1):290. DOI:10.1186/s12917-014-0290-8 · 1.74 Impact Factor
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ABSTRACT: Thiopurine drugs are widely used as anti-leukemic drugs and immunosuppressive agents, and 6-thioguanosine triphosphate ((S)GTP) is a major metabolite for these drugs. Recent studies suggested that thiopurine drugs may exert their cytotoxic effects partly through binding of (S)GTP to a GTP-binding protein, Rac1. However, it remains unclear whether (S)GTP can also bind to other cellular proteins. Here, we introduced an orthogonal approach, encompassing nucleotide-affinity profiling and nucleotide-binding competition assays, to characterize comprehensively (S)GTP-binding proteins along with the specific binding sites from the entire human proteome. With the simultaneous use of (S)GTP and GTP affinity probes, we identified 165 (S)GTP-binding proteins that are involved in several different biological processes. We also examined the binding selectivities of these proteins toward (S)GTP and GTP, which allowed for the revelation of the relative binding affinities of the two nucleotides toward the nucleotide-binding motif sequence of proteins. Our results suggest that (S)GTP mainly targets GTPases, with strong binding affinities observed for multiple heterotrimeric G proteins. We also demonstrated that (S)GTP binds to several cyclin-dependent kinases (CDKs), which may perturb the CDK-mediated phosphorylation and cell cycle progression. Together, this represents the first comprehensive characterization of (S)GTP-binding property for the entire human proteome. We reason that a similar strategy can be generally employed for the future characterization of the interaction of other modified nucleotides with the global proteome.Analytical Chemistry 04/2014; 86(9). DOI:10.1021/ac500588q · 5.83 Impact Factor
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ABSTRACT: Indoleamine 2,3-dioxygenase (IDO) is found in multiple malignancies and exerts immunosuppressive effects that are central in protecting tumors from host T lymphocyte rejection. IDO is an enzyme involved in the catabolism of tryptophan resulting in inhibition of T lymphocyte function. While inhibition of IDO enzymatic activity results in tumor rejection, it is still unknown how we can directly target IDO expression within tumors using drugs. We have chosen to interfere with IDO expression by targeting the key-signaling event signal transducer and activator of transcription 1 (STAT1). We evaluated the efficacy of fludarabine, previously described to inhibit STAT1 phosphorylation. Interestingly, fludarabine was efficient in suppressing protein expression and consequently IDO activity in two different cell lines derived from breast cancer and melanoma when IDO was activated with interferon-gamma (IFN-γ) or supernatants prepared from activated T lymphocytes. However, fludarabine had no inhibitory effect on STAT1 phosphorylation. Other IFN-γ-responsive genes were only marginally inhibited by fludarabine. The level of IDO transcript was unaffected by this inhibitor, suggesting the involvement of post-transcriptional control. Strikingly, we have found that the inhibition of proteasome partially protected IDO from fludarabine-induced degradation, indicating that fludarabine induces IDO degradation through a proteasome-dependent pathway. Currently used in the clinic to treat some malignancies, fludarabine has the potential for use in the treatment of human tumors through induction of IDO degradation and consequently, for the promotion of T cell-mediated anti-tumor response.PLoS ONE 06/2014; 9(6):e99211. DOI:10.1371/journal.pone.0099211 · 3.53 Impact Factor