Knockdown of μ-Calpain in Fanconi Anemia, FA-A, Cells by siRNA Restores αII Spectrin Levels and Corrects Chromosomal Instability and Defective DNA Interstrand Cross-Link Repair

We have previously shown that there is a deficiency in the structural protein, nonerythroid alpha spectrin (alpha IISp), in cells from patients with Fanconi anemia (FA). These studies indicate that this deficiency is due to the reduced stability of alpha IISp and correlates with a decreased level of repair of DNA interstrand cross-links and chromosomal instability in FA cells. An important factor in the stability of alpha IISp is its susceptibility to cleavage by the protease, mu-calpain. We hypothesized that an increased level of mu-calpain cleavage of alpha IISp in FA cells leads to an increased level of breakdown of alpha IISp and that knocking down expression of mu-calpain in FA cells should restore levels of alpha IISp and correct a number of the phenotypic defects observed. The results showed that there is increased mu-calpain activity in FA-A, FA-C, FA-D2, FA-F, and FA-G cells that could account for the deficiency in alpha IISp in these FA cells. Protein interaction studies indicated that FANCA and FANCG bind directly to mu-calpain. We hypothesize that this binding may lead to inhibition of mu-calpain activity in normal cells. Knocking down mu-calpain by siRNA in FA-A cells restored levels of alpha IISp to normal and reversed a number of the cellular deficiencies in these cells. It corrected the DNA repair defect and the chromosomal instability observed after exposure to a DNA interstrand cross-linking agent. These studies indicate that FA proteins may play an important role in maintaining the stability of alpha IISp in the cell by regulating its cleavage by mu-calpain. Thus, by reducing the level of breakdown of alpha IISp in FA cells, we may be able to reverse a number of the cellular deficiencies observed in this disorder.