Chromatin negative nuclei
Chromatin negative nuclei free#
The total and free drug concentrations (C t and C f, respectively) were determined using drug extinction coefficient of 19 200 M -1 cm -1 and the amount of bound drug (C b) was obtained from C b = C t –C f.
Chromatin negative nuclei serial#
Serial concentrations of mitoxantrone was made in the same buffer and used as a control. The samples were then eppendorfed and the absorbance of the supernatants was measured at multi λ system using Shimadzo UV-160 spectrophotometer, equipped with quartz cells at constant temperature. The nuclei suspension and soluble chromatin were diluted with the reaction buffer (10 mM Tris –HCl pH 7.4 or 0.25 M sucrose, 25 mM NaCl and 10 mM Tris-HCl, pH 7.4) to the final concentration of 100-200 μg/ml DNA and then incubated with various concentrations of mitoxantrone for 45 min at room temperature in the dark. The results suggest release of proteins upon drug binding to nuclei and linker region are introduced as a preferable sites for mitoxantrone interaction. In the present study, we have attempted to characterize the binding of anticancer drug mitoxantrone to intact nuclei and compared to soluble chromatin with especial emphasize on chromatin proteins. We have previously shown that mitoxantrone binds to chromatin with higher affinity compared to DNA, implying that the histone proteins may play an important role in the chromatin- mitoxantrone interaction process. In the cell, chromatin has been introduced as a tool for study of genome function in cancer.
They bind to DNA and chromatin and act as “architectural elements” that induce both short- and long-range changes in the structure of their binding sites and facilitate various DNA-related activities. The high mobility group (HMG) proteins are a small set of non histone proteins with relatively low molecular weights and high solubility in water. Histone H1 is a very lysine-rich histone fraction of chromatin, which binds to linker DNA between adjacent nucleosomes to facilitate the folding of the chromatin fiber. They are H2A, H2B, H3, and H4, which associate as H2A/H2B dimers and H3/H4 tetramer, arranged in an octamer form. The core histones are small, basic proteins with more than 50% of their amino acid composition is lysine and arginine.
There are five main histones in the cell nucleus: the linker histones of the H1 family and four core or nucleosomal histones. In eukaryotes, DNA is complexed with histones and other nuclear proteins producing a defined structure known as chromatin. Mitoxantrone also belongs to the class of topoisomerase II inhibitor. Numerous studies on the interaction of mitoxantrone with DNA have been undertaken and all indicate that the drug exerts its biological function via intercalation into DNA double strands. © 2014 The Authors Developmental Neurobiology Published by Wiley Periodicals, Inc.Mitoxantrone is a synthetic antibiotic widely used as a chemotherapeutic drug for the treatment of solid tumors, leukemia, and lymphoma.
The further analysis of decondensed neural cell nuclei should provide novel insights into neurobiology and neurodegenerative disease.Ĭell-cycle cytometry epigenetics nuclei pluripotency sorting. NeuN-High nuclei have the properties consistent with their being derived from extremely active neurons with elevated rates of chromatin modification and/or NPC-like cells with multilineage developmental potential. The cortex, hippocampus, hypothalamus, thalamus, and nucleus accumbens contained high percentages of large decondensed NeuN-High nuclei, while the cerebellum, and pons contained very few. NeuN-High nuclei expressed higher levels of HDAC1, 2, 4, and 5 proteins. Purified NeuN-High nuclei expressed significantly higher levels of transcripts encoding markers of neurogenesis, neuroplasticity, and learning and memory (ARC, BDNF, ERG1, HOMER1, NFL/NEF1, SYT1), subunits of chromatin modifying machinery (SIRT1, HDAC1, HDAC2, HDAC11, KAT2B, KAT3A, KAT3B, KAT5, DMNT1, DNMT3A, Gadd45a, Gadd45b) and markers of NPC and cell cycle activity (BRN2, FOXG1, KLF4, c-MYC, OCT4, PCNA, SHH, SOX2) relative to neuronal NeuN-Low or to mostly non-neuronal NeuN-Neg nuclei. We found that mouse brain cell nuclei that expressed exceptionally high levels of the pan neuronal marker NeuN/FOX3 (NeuN-High) had decondensed chromatin relative to most NeuN-Low or NeuN-Neg (negative) nuclei.
Although multipotent cell types have enlarged nuclei with decondensed chromatin, this property has not been exploited to enhance the characterization of neural progenitor cell (NPC) populations in the brain.