Expression and significance of DNA topoisomerase II in neural stem cells of ICR mice

doi:10.12307/2022.014

Abstract

Abstract: BACKGROUND: Embryonic neural stem cells can differentiate into neurons and glial cells. In practical applications, we hope that neural stem cells will differentiate into neurons more. Therefore, it is very important to study the mechanism of directional differentiation of neural stem cells into neurons. DNA topoisomerase II is a type of nucleoprotein widely present in organisms and plays an important role in life activities such as DNA replication, repair, transcription, recombination and chromosome separation.
OBJECTIVE: To explore the expression and significance of DNA topoisomerase II in neural stem cells and their directional differentiation into neurons.
METHODS: Neural stem cells were isolated from cerebral cortex of ICR mice (E12.5 d) and cultured. The single cells after digestion were inoculated in a petri dish at 5×108 L-1 after matting with Matrigel. The conditioned medium of neurogenic differentiation containing B27, N2 and 10 μg/L glial cell line derived neurotrophic factor was added. At 1, 2, and 3 days after culture, the expression of DNA topoisomerase II α and β was detected by immunocytochemistry, western blot assay, and real time-quantitative PCR.
RESULTS AND CONCLUSION: (1) The number of DNA topoisomerase IIα-positive cells in neurospheres was large and evenly distributed, and the number of positive cells after induced differentiation was reduced. The number of DNA topoisomerase IIβ-positive cells in neurospheres was relatively small, mainly distributed in the center of neurospheres. After induced differentiation, the number of positive cells began to increase gradually. At 2 days, the expression reached a peak, and then showed a decreasing trend. (2) The expression of DNA topoisomerase IIα protein and mRNA in neurospheres was high and gradually decreased after the differentiation into neurons. The expression of DNA topoisomerase IIβ protein and mRNA in neurospheres was low and significantly increased after neuronal differentiation. (3) Through experiments, it is concluded that DNA topoisomerase IIβ participates in the directional differentiation of neural stem cells into neurons.

Key words: neural stem cells, DNA topoisomerase II α, DNA topoisomerase II β, immunocytochemistry, western blot, real time-quantitative PCR

CLC Number:

Sun Mengjun, Guo Jianmei, Zhou Gengsu, Dong Zefei, Zheng Chungui, Cao Cuili. Expression and significance of DNA topoisomerase II in neural stem cells of ICR mice[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(1): 84-89.

Figures/Tables 5

References

[1] THAN-TRONG E, ORTICA-GATTI S, MELLA S, et al. Neural stem cell quiescence and stemness are molecularly distinct outputs of the Notch3 signalling cascade in the vertebrate adult brain. Development. 2018;145(10):dev161034.
[2] MOORE L, SKOP NB, ROTHBARD DE, et al. Tethered growth factors on biocompatible scaffolds improve stemness of cultured rat and human neural stem cells and growth of oligodendrocyte progenitors. Methods. 2018;133:54-64.
[3] FISCHER U, BACKES C, RASLAN A, et al. Gene amplification during differentiation of mammalian neural stem cells in vitro and in vivo. Oncotarget. 2015;6(9):7023-7039.
[4] COSACAK MI, BHATTARAI P, KIZIL C. Alzheimer’s disease, neural stem cells and neurogenesis: cellular phase at single-cell level. Neural Regen Res. 2020;15(5):824-827.
[5] DRAKE FH, ZIMMERMAN JP, MCCABE FL, et al. Purification of topoisomerase II from amsacrine-resistant P388 leukemia cells. Evidence for two forms of the enzyme. J Biol Chem. 1987;262(34): 16739-16747.
[6] TSAI-PFLUGFELDER M, LIU LF, LIU AA, et al. Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22. Proc Natl Acad Sci U S A. 1988;85(19):7177-7181.
[7] JENKINS JR, AYTON P, JONES T, et al. Isolation of cDNA clones encoding the beta isozyme of human DNA topoisomerase II and localisation of the gene to chromosome 3p24. Nucleic Acids Res. 1992;20(21): 5587-5592.
[8] SOCZEK KM, GRANT T, ROSENTHAL PB, et al. CryoEM structures of open dimers of gyrase A in complex with DNA illuminate mechanism of strand passage. Elife. 2018;7:e41215.
[9] YANG X, LI W, PRESCOTT ED, et al. DNA topoisomerase IIbeta and neural development. Science. 2000;287(5450):131-134.
[10] TSUTSUI K, TSUTSUI K, SANO K, et al. Involvement of DNA topoisomerase IIbeta in neuronal differentiation. J Biol Chem. 2001; 276(8):5769-5778.
[11] LYU YL, WANG JC. Aberrant lamination in the cerebral cortex of mouse embryos lacking DNA topoisomerase IIbeta. Proc Natl Acad Sci U S A. 2003;100(12):7123-7128.
[12] 孙孟军,郭建美,周更苏,等. ICR小鼠神经干细胞的体外培养与鉴定[J].河北医科大学学报, 2019, 40(3):258-263.
[13] 孙孟军,郭建美,周更苏,等. ICR小鼠神经干细胞的诱导分化及鉴定[J].医学动物防制, 2021, 37(5):467-470.

[14] BAKSHI RP, GALANDE S, MUNIYAPPA K. Functional and regulatory characteristics of eukaryotic type II DNA topoisomerase. Crit Rev Biochem Mol Biol. 2001;36(1):1-37.
[15] LEE JH, BERGER JM. Cell Cycle-Dependent Control and Roles of DNA Topoisomerase II. Genes (Basel). 2019;10(11):859.
[16] TORRES-GUZMÁN R, CALSINA B, HERMOSO A, et al. Preclinical characterization of abemaciclib in hormone receptor positive breast cancer. Oncotarget. 2017;8(41):69493-69507.
[17] MOROTOMI-YANO K, SAITO S, ADACHI N, et al. Dynamic behavior of DNA topoisomerase IIβ in response to DNA double-strand breaks. Sci Rep. 2018;8(1):10344.
[18] MILLS WE, SPENCE JM, FUKAGAWA T, et al. Site-Specific Cleavage by Topoisomerase 2: A Mark of the Core Centromere. Int J Mol Sci. 2018;19(2):534.
[19] JOSHI RS, NIKOLAOU C, ROCA J. Structure and Chromosomal Organization of Yeast Genes Regulated by Topoisomerase II. Int J Mol Sci. 2018;19(1):134.
[20] GOTHE HJ, BOUWMAN BAM, GUSMAO EG, et al. Spatial Chromosome Folding and Active Transcription Drive DNA Fragility and Formation of Oncogenic MLL Translocations. Mol Cell. 2019;75(2):267-283.e12.
[21] MADABHUSHI R. The Roles of DNA Topoisomerase IIbeta in Transcription. Int J Mol Sci. 2018;19(7):1917.
[22] BALDYS-WALIGORSKA A, WIERZBICKA I, SOKOLOWSKI G, et al. Markers of proliferation and invasiveness in somatotropinomas. Endokrynol Pol. 2018;69(2):182-189.
[23] TERZIOGLU-USAK S, NEGIS Y, KARABULUT DS, et al. Cellular Model of Alzheimer’s Disease: Aβ1-42 Peptide Induces Amyloid Deposition and a Decrease in Topo Isomerase IIβ and Nurr1 Expression. Curr Alzheimer Res. 2017;14(6):636-644.
[24] TSUTSUI K, TSUTSUI KM, MIYAJI M, et al. Regulation of neuronal gene expression by DNA topoisomerase IIbeta. Tanpakushitsu Kakusan Koso. 2009;54(11):1333-1343.
[25] TSUTSUI K, TSUTSUI K, SAKURAI H, et al. Levels of topoisomerase II and DNA polymerase alpha are regulated independently in developing neuronal nuclei. Biochem Biophys Res Commun. 1986;138(3):1116-1122.
[26] TSUTSUI K, TSUTSUI K, HOSOYA O, et al. Immunohistochemical analyses of DNA topoisomerase II isoforms in developing rat cerebellum. J Comp Neurol. 2001;431(2):228-239.
[27] WATANABE M, TSUTSUI K, TSUTSUI K, et al. Differential expressions of the topoisomerase II alpha and II beta mRNAs in developing rat brain. Neurosci Res. 1994;19(1):51-57.
[28] TSUTSUI KM, SANO K, HOSOYA O, et al. Expression dynamics and functional implications of DNA topoisomerase II beta in the brain. Anat Sci Int. 2006;81(3):156-163.
[29] KONDAPI AK, MULPURI N, MANDRAJU RK, et al. Analysis of age dependent changes of Topoisomerase II alpha and beta in rat brain. Int J Dev Neurosci. 2004;22(1):19-30.
[30] BHANOTHU V, KONDAPI AK. Status of topoisomerase-2β protein in all-trans retinoic acid-treated human neuroblastoma (SK-N-SH) cells. J Cell Biochem. 2019;120(4):5169-5182.
[31] ZAIM M, ISIK S. DNA topoisomerase IIβ stimulates neurite outgrowth in neural differentiated human mesenchymal stem cells through regulation of Rho-GTPases (RhoA/Rock2 pathway) and Nurr1 expression. Stem Cell Res Ther. 2018;9(1):114.
[32] NUR-E-KAMAL A, MEINERS S, AHMED I, et al. Role of DNA topoisomerase IIbeta in neurite outgrowth. Brain Res. 2007;1154: 50-60.
[33] MAEDA Y, TSUTSUI K, TOKUNAGA A. Regional differences in the expression of DNA topoisomerase IIbeta in the pyramidal neurons of the rat hippocampus. Neurosci Res. 2000;36(4):291-296.