Knockdown of αII spectrin in normal human cells by siRNA leads to chromosomal instability and decreased DNA interstrand cross-link repair

Abstract

Nonerythroid α-spectrin (αIISp) is a structural protein involved in repair of DNA interstrand crosslinks and is deficient in cells from patients with Fanconi anemia (FA), which are defective in ability to repair cross-links. In order to further demonstrate the importance of the role that αIISp plays in normal human cells and in the repair defect in FA, αIISp was knocked down in normal cells using siRNA. Depletion of αIISp in normal cells by siRNA resulted in chromosomal instability and cellular hypersensitivity to DNA interstrand cross-linking agents. An increased number of chromosomal aberrations were observed and, following treatment with a DNA interstrand cross-linking agent, mitomycin C, cells showed decreased cell growth and survival and decreased formation of damage induced αIISp and XPF nuclear foci. Thus depletion of αIISp in normal cells leads to a number of defects observed in FA cells, such as chromosome instability and a deficiency in cross-link repair. Keywordsα-spectrin; siRNA knockdown; Chromosomal aberrations; DNA interstrand crosslinks; Damageinduced nuclear foci; XPF; DNA repair; Fanconi anemia Spectrin is a structural protein, which in the cytoplasm of nonerythroid cells plays an important role in providing mechanical support for the cell membrane and is also involved in protein sorting, organelle and vesicle trafficking, neurite outgrowth and neurotransmitter release, as well as cell growth and differentiation [1][2][3][4]. We have demonstrated that nonerythroid α-spectrin (αIISp) is present in mammalian cell nuclei and is involved in repair of DNA interstrand cross-links [5][6][7]. It preferentially binds to purified DNA containing an interstrand cross-link; it co-localizes with the cross-link repair protein, XPF, and the Fanconi anemia protein, FANCA, in cross-link induced nuclear foci, and it is needed for the production of incisions produced by XPF at the site of an interstrand cross-link [5][6][7].

Serum Glial Fibrillary Acidic Protein Predicts Tissue Glial Fibrillary Acidic Protein Break-Down Products and Therapeutic Efficacy after Penetrating Ballistic-Like Brain Injury

“…However, studies have shown that protection or recovery of α-II spectrin is correlated to neuronal survival 43 . Spectrin is also involved in repairing DNA damage 44 , a process which occurs in TBI 45,46 . Perhaps the increase in α-II spectrin 7 d after injury is an indicator of spontaneous repair.…”

αII Spectrin Stabilizes Stress Fibers and Actin–Membrane Interactions

“…Previous reports of siRNA knockdown suggest that complete knockdown of aII-spectrin is lethal to cells. 19 We used antibiotic selection to maintain the aII-spectrin reduced phenotype and prevent the overgrowth of untransfected cells.…”

Multiple roles of ERCC1‐XPF in mammalian interstrand crosslink repair

“…Additionally, aIISp colocalizes with FANCA and XPF forming discrete foci in response to 8-MOP plus UVA light, indicating a possible functional link between these proteins in response to ICL damage [Sridharan et al, 2003]. Knockdown of aIISp resulted in formation of fewer nuclear foci following treatment with MMC as well as decreased survival, further implicating this protein in ICL recognition [McMahon et al, 2009]. A consequence of this damage recognition is recruitment of repair proteins including ERCC1-XPF to the site of DNA damage in preparation for the next step of the repair process.…”