BackgroundThe cytidine nucleoside analogs azacitidine (AZA) and decitabine (DAC) are used for the treatment of patients with myelodysplastic syndromes and acute myeloid leukemia (AML). Few non-clinical studies have directly compared the mechanisms of action of these agents in a head-to-head fashion, and the agents are often viewed as mechanistically similar DNA hypomethylating agents. To better understand the similarities and differences in mechanisms of these drugs, we compared their in vitro effects on several end points in human AML cell lines.Methodology/Principal FindingsBoth drugs effected DNA methyltransferase 1 depletion, DNA hypomethylation, and DNA damage induction, with DAC showing equivalent activity at concentrations 2- to 10-fold lower than AZA. At concentrations above 1 µM, AZA had a greater effect than DAC on reducing cell viability. Both drugs increased the sub-G1 fraction and apoptosis markers, with AZA decreasing all cell cycle phases and DAC causing an increase in G2-M. Total protein synthesis was reduced only by AZA, and drug-modulated gene expression profiles were largely non-overlapping.Conclusions/SignificanceThese data demonstrate shared mechanisms of action of AZA and DAC on DNA-mediated markers of activity, but distinctly different effects in their actions on cell viability, protein synthesis, cell cycle, and gene expression. The differential effects of AZA may be mediated by RNA incorporation, as the distribution of AZA in nucleic acid of KG-1a cells was 65∶35, RNA∶DNA.
Excitatory synaptic activity can evoke transient and substantial elevations of postsynaptic calcium. Downstream effects of elevated calcium include the activation of the calcium-dependent protease calpain. We have developed a reagent that identifies dendritic spines in which calpain has been activated. A fusion protein was expressed that contained enhanced yellow and enhanced cyan fluorescent protein (EYFP and ECFP, respectively) linked by a peptide that included the -calpain cleavage site from ␣-spectrin. A PDZ-binding site fused to ECFP anchored this protein to postsynaptic densities. The fusion protein exhibited fluorescence resonance energy transfer (FRET), and diminution of FRET by proteolysis was used to localize calpain activity in situ by fluorescence microscopy. Incubation of the fusion protein with calpain in the presence of calcium resulted in the separation of EYFP and ECFP into monomeric fluorophores. In transiently transfected cell lines and dissociated hippocampal neurons, FRET was diminished by raising intracellular calcium levels with an ionophore or with glutamatergic agonists. Calpain inhibitors blocked these changes. Under control conditions, FRET levels in different dendritic spines of cultured neurons and in hippocampal slices were heterogeneous but showed robust decreases upon treatment with glutamatergic agonists. Immunostaining of cultured neurons with antibodies to a spectrin epitope produced by calpain-mediated digestion revealed an inverse correlation between the amount of FRET present at postsynaptic elements and the concentration of spectrin breakdown products. These results suggest that the FRET methodology identifies sites of synaptically induced calpain activity and that it may be useful in analyzing synapses undergoing changes in efficacy.A ctivity-dependent increases in synaptic efficacy are thought to be necessary for several forms of learning and memory (for review, see refs. 1-3). A critical event for the induction of stable changes in synaptic strength appears to be a large but transient increase in intracellular calcium (4, 5). Attempts to understand the molecular and cellular mechanisms underlying synaptic plasticity have been limited by an inability to resolve functional changes of individual synapses at a histological level. Although recent reports have demonstrated biochemical and morphological alterations in response to localized manipulations of synaptic activity (6-8), most studies rely on sampling methods that cannot discriminate between synaptic sites that have undergone functional change and the majority of the population, which remains unchanged. It therefore would be useful to have an enzymatic reporter to mark individual synapses that have undergone functional change. A useful marker enzyme should be dependent on the levels of calcium required for synaptic plasticity, have a low background activation, and have substrates that are not equivalently modified by other enzymes. The calciumdependent protease -calpain satisfies all the above criteria (9). Calpain is ...
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