e-3330 and methoxyamine

Apurinic/apyrimidinic endonuclease 1 (APE1) is the predominant mammalian enzyme in DNA base excision repair pathway that cleaves the DNA backbone immediately 5' to abasic sites. In addition to its abasic endonuclease activity, APE1 has 3' phosphatase and 3'-5' exonuclease activities against DNA. We recently identified APE1 as an endoribonuclease that preferentially cleaves at UA, UG, and CA sites in single-stranded regions of RNAs and can regulate c-myc mRNA level and half-life in cells. APE1 can also endonucleolytically cleave abasic single-stranded RNA. Here, we show for the first time that the human APE1 has 3' RNA phosphatase and 3' exoribonuclease activities. Using three distinct RNA substrates, we show that APE1, but not RNase A, can remove the phosphoryl group from the 3' end of RNA decay products. Studies using various site-directed APE1 mutant proteins (H309N, H309S, D283N, N68A, D210N, Y171F, D308A, F266A, and D70A) suggest that the 3' RNA phosphatase activity shares the same active center as its other known nuclease activities. A number of APE1 variants previously identified in the human population, including the most common D148E variant, have greater than 80% reduction in the 3' RNA phosphatase activity. APE1 can remove a ribonucleotide from the 3' overhang of RNA decay product, but its 3'-5' exoribonuclease activity against unstructured poly(A), poly(C), and poly(U) RNAs is relatively weak. This study further underscores the significance of understanding the role of APE1 in RNA metabolism in vivo.

Topics: Benzoquinones; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Endoribonucleases; Exoribonucleases; Flavonoids; Half-Life; Humans; Hydroxylamines; Phosphoric Monoester Hydrolases; Propionates; RNA; Substrate Specificity

Soy isoflavones sensitize cancer cells to radiation both in vitro and in vivo. To improve the effect of radiotherapy for non-small cell lung cancer, we assessed the potential of using a complementary approach with soy isoflavones.. Human A549 non-small cell lung cancer cells were treated with soy isoflavones, radiation, or both and tested for cell growth. DNA double-strand breaks (DSBs) were detected by immunostaining for γ-H2AX foci. Expressions of γ-H2AX, HIF-1α, and APE1/Ref-1 were assessed by Western blots. DNA-binding activities of HIF-1α and NF-κB transcription factors were analyzed by electrophoretic mobility shift assay.. Soy isoflavones increased A549 cell killing induced by radiation. Multiple γ-H2AX foci were detectable at 1 hour after radiation but decreased at 24 hours after radiation. Soy isoflavones also caused DNA DSBs, but γ-H2AX foci increased over time. Soy isoflavones and radiation caused an increase in γ-H2AX foci, which persisted at 24 hours, indicating both increased DNA damage and inhibition of repair. Soy isoflavones inhibited the radiation-induced activity of the DNA repair/redox enzyme APE1/Ref-1 and the transcription factors NF-κB and HIF-1α. E3330, which inhibits the redox activity of APE1/Ref-1, did not alter the repair of radiation-induced DSBs. Methoxyamine, which inhibits APE1/Ref-1 DNA repair activity, partly blocked the decrease in radiation-induced DSBs at 24 hours, suggesting partial mitigation of radiation-induced DNA repair akin to the effect of soy combined with radiation, in agreement with cytotoxic assays.. Inhibition of APE1/Ref-1 DNA repair activity by soy could be involved in the mechanism by which soy alters DNA repair and leads to cell killing.

Topics: Benzoquinones; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; DNA Damage; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Electrophoretic Mobility Shift Assay; Fluorescent Antibody Technique; Glycine max; Histones; Humans; Hydroxylamines; Hypoxia-Inducible Factor 1, alpha Subunit; Isoflavones; Lung Neoplasms; NF-kappa B; Propionates; Tumor Cells, Cultured; X-Rays

Ape1 is a molecule with dual functions in DNA repair and redox regulation of transcription factors. In Ape1-deficient mice, embryos do not survive beyond embryonic day 9, indicating that this molecule is required for normal embryo development. Currently, direct evidence of the role of Ape1 in regulating hematopoiesis is lacking. We used the embryonic stem (ES) cell differentiation system and an siRNA approach to knockdown Ape1 gene expression to test the role of Ape1 in hematopoiesis. Hemangioblast development from ES cells was reduced 2- to 3-fold when Ape1 gene expression was knocked down by Ape1-specific siRNA, as was primitive and definitive hematopoiesis. Impaired hematopoiesis was not associated with increased apoptosis in siRNA-treated cells. To begin to explore the mechanism whereby Ape1 regulates hematopoiesis, we found that inhibition of the redox activity of Ape1 with E3330, a specific Ape1 redox inhibitor, but not Ape1 DNA repair activity, which was blocked using the small molecule methoxyamine, affected cytokine-mediated hemangioblast development in vitro. In summary, these data indicate Ape1 is required in normal embryonic hematopoiesis and that the redox function, but not the repair endonuclease activity, of Ape1 is critical in normal embryonic hematopoietic development.

Topics: Animals; Apoptosis; Benzoquinones; Cell Differentiation; Cells, Cultured; DNA; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Embryonic Stem Cells; Gene Expression; Hematopoiesis; Hydroxylamines; Mice; Oxidation-Reduction; Propionates; RNA, Small Interfering