Abstract
Abstract 495
Fanconi Anemia (FA) is a rare recessive chromosomal-instability disorder characterized by congenital malformations, a high predisposition to cancer, and progressive bone marrow failure. FA is genetically heterogeneous and, to date, thirteen FA genes have been identified (FANCA, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N). The thirteen encoded FA proteins cooperate in a common DNA repair pathway active during the Synthesis (S) phase of the cell cycle. DNA damage detected during replication results in the monoubiquitination of two FA proteins, FANCD2 and FANCI, that translocate into chromatin-associated DNA repair foci where they colocalize with downstream components of the pathway. Partial colocalization with BLM, the RecQ helicase mutated in Bloom's syndrome, has also been described. How disruption of this pathway leads to bone marrow failure is a critical unanswered question. Interestingly, FA cells also have abnormalities that suggest a defect in mitosis, including micronuclei and multinucleation. The objectives of this study were to 1) investigate the role of the FA pathway in normal mitosis and 2) determine whether defects in this function underlie the bone marrow failure of FA patients. For this study, we used HeLa cells transiently or stably knocked down for FA genes, FA patient derived cell lines and hematopoietic stem cells from Fanconi mice models generated in our laboratory (Fancd2-/- and Fancg-/-). First, a polyclonal antibody was raised against FANCI and, together with an anti-FANCD2 antibody, used to investigate the localization of the FANCD2-I complex throughout the cell cycle by immunostaining. FANCI and FANCD2 colocalized to discrete foci on condensed chromosomes in a population of cells in Mitosis (M) phase, consistent with results of Chan et al. (Replication stress induces sister-chromatid bridging at fragile site loci in mitosis. Nat Cell Biol. 2009;11:753-760), Naim and Rosselli (The FANC pathway and BLM collaborate during mitosis to prevent micro-nucleation and chromosome abnormalities. Nat Cell Biol. 2009;11:761-768). These foci were dependent on an intact FA pathway, but did not localize at centromeres and did not increase when the spindle assembly checkpoint was challenged. By immunofluorescence, we showed an increase in the presence of Hoechst positive DNA bridges and PICH positive / BLM positive DNA bridges (Hoechst positive and negative) in anaphase and telophase of FA deficient cells compared to FA proficient cells. This increase of DNA bridges between separating sister chromatids in FA deficient cells correlated with an increase of multinucleated cells. Multinuclearity, scored by immunostaining for microtubules and Hoechst staining for DNA, was the result of cytokinesis failure as observed by live cell imaging. Furthermore, inhibition of apoptosis increased the number of binucleated cells, suggesting that cytokinesis failure led to apoptosis. Importantly, an increase in binucleated cells was also observed in the hematopoietic stem cells population from Fancd2-/- and Fancg-/- mice, compared to wild-type sibling mice, and this increase correlated with elevated apoptosis in those cells. Based on these new findings, we conclude that the Fanconi pathway is required for normal mitosis and hypothesize that apoptosis induced by cytokinesis failure of hematopoietic stem cells may cause the bone marrow failure commonly found in FA patients.
Disclosures:
No relevant conflicts of interest to declare.
Faculty Opinions recommendation of Bone marrow failure in Fanconi anemia is triggered by an exacerbated p53/p21 DNA damage response that impairs hematopoietic stem and progenitor cells.
