Restoring Levels of αII Spectrin in Fanconi Anemia Group A Cells Leads to Reversal of the Chromosomal Instability and to Enhanced Ability to Repair DNA Interstrand Cross-Links.

Abstract 493

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, a predisposition to cancer, congenital abnormalities, genomic instability, and a defect in ability to repair DNA interstrand cross-links. We have shown that the structural protein, nonerythroid a spectrin (aIISp), plays an important role in the repair of DNA interstrand cross-links and that there is a deficiency in aIISp in FA cells. We have recently demonstrated that aIISp is also important in chromosome stability and that its depletion in normal cells by siRNA, to levels found in FA cells, leads to a number of the defects observed in FA cells, such as increased chromosomal instability, increased cellular sensitivity to DNA interstrand cross-linking agents and decreased ability to repair DNA interstrand cross-links. We have shown that the reduced levels of αIISp in FA cells are due to its increased breakdown. The goal of the present study was to determine whether, by increasing the levels of aIISp in FA cells to normal, it would be possible to enhance chromosome stability and decrease the sensitivity to DNA interstrand cross-linking agents and the DNA repair defect. In the cell, aIISp is cleaved by the protease, m calpain, to give distinctive cleavage products. Cells from a number of FA complementation groups were examined for levels and activity of m calpain. The results showed that there was increased m calpain activity in FA cells, which included FA-A, FA-C, FA-D2, FA-F and FA-G, which correlated increased m calpain breakdown product of αIISp in these cells. However, levels of calpain in the FA cells were similar to normal. In FA cells corrected with the corresponding FANC cDNA, activity of m calpain was reduced to normal levels and αIISp levels returned to normal, indicating a role for the FA proteins in regulating calpain activity and αIISp stability. Studies were carried out to determine whether FA proteins bound directly to calpain. Co-immunoprecipitation and yeast two-hybrid analysis showed that FANCA and FANCG bound directly to calpain and thus could potentially play a role in its activity. Studies were also undertaken to determine whether knockdown of m calpain in FA cells by siRNA could lead to restoration of αIISp levels and reversal of some of the cellular phenotypic characteristics of FA. The results showed that when m calpain was knocked down in FA-A cells by siRNA, αIISp levels returned to normal. When these siRNA transfected FA-A cells were exposed to a DNA interstrand cross-linking agent, mitomycin C (MMC), cell survival increased to ∼85% of normal. After treatment of these cells with MMC, αIISp and a cross-link repair protein, XPF, nuclear foci were observed at levels similar to normal. This is in contrast to nontarget transfected FA-A cells in which few damage-induced αIISp or XPF nuclear foci were seen. In addition, in cells in which m calpain was knocked down, chromosomal stability in these cells was enhanced. Few of the chromosomal abnormalities usually observed in FA-A cells after MMC treatment, such as chromosome breaks and rearrangements, were observed. These studies thus indicate that increased calpain activity in FA cells could be responsible for decreased αIISp in these cells. Several of the FA proteins could play an important role in regulating activity of m calpain and thus in maintaining normal levels of αIISp, which in turn is critical for maintenance of chromosome stability and DNA repair. Knocking down m calpain in FA-A cells restores levels of αIISp to normal and enhances chromosome stability and DNA repair in these cells. This could potentially have important therapeutic relevance.