siRNA-Mediated Silencing of αII Spectrin Gene Expression in Cells Containing DNA Interstrand Cross-Links and Its Relationship to Fanconi Anemia.

Fanconi anemia (FA) is characterized by bone marrow failure, a predisposition to cancer and, at the cellular level, a hypersensitivity to DNA interstrand cross-linking agents that correlates with a defect in ability to repair cross-links produced by these agents. We have previously shown that the structural protein, nonerythroid a spectrin (αSpII∑*), binds to DNA containing interstrand cross-links and plays an important role in the repair of this type of damage and that there is a deficiency in this protein in cells from FA-A, FA-B, FA-C, FA-D1, FA-F and FA-G cells. We have also shown that after damage to normal cells, αSpII∑* co-localizes in nuclear foci with FANCA, FANCF and the DNA interstrand cross-link repair protein XPF. In FA-A cells, where there is a deficiency in αSpII∑*, damage-induced nuclear foci formation is significantly reduced. In order to further assess the functional importance of αSpII∑* in the repair process and the DNA repair defect in FA cells, studies were carried out on siRNA-mediated silencing of αII spectrin gene expression. Three siRNA oligonucleotide templates were synthesized, which were targeted for different αII spectrin gene sequences. The 21-mer siRNAs produced were purified and normal human lymphoblastoid cells were transfected with these siRNAs. αII spectrin mRNA and protein levels in transfected cells were determined by RT-PCR and Western blot analysis, respectively. By 48 hours after transfection, levels of αII spectrin mRNA and protein were approximately 34% and 30% of mock transfected cells, respectively. Cells were then damaged with 8-MOP plus UVA light so as to produce DNA interstrand cross-links and localization of αII spectrin and FANCA in the nuclei was examined by immunofluorescence microscopy. The results showed that, in cells transfected with αII spectrin siRNA, there was a marked reduction in the number of αII spectrin and FANCA foci in the nuclei of the siRNA transfected cells compared to the mock transfected cells. There was also markedly reduced survival of the siRNA transfected cells after damage compared to mock transfected cells. These results show that there is a correlation between a reduction of αII spectrin levels in these cells and decreased formation of FANCA as well as αII spectrin nuclear foci after damage with a DNA interstrand cross-linking agent and that this in turn correlates with decreased survival and DNA repair in these cells after DNA damage. These results support our model that αII spectrin is needed in the DNA repair process where it acts as a scaffold in the recruitment and alignment of FANC and repair proteins at sites of DNA damage and that, in FA cells, where there is a deficiency in αII spectrin, this recruitment and repair are defective.

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