DNA Cross-Linking Agent Sensitivity of Fanconi Anemia (FA) Cells Is Preserved in Double Knockout (DKO) SMAD3^-/- Fancd2^-/- Mouse Cell Lines

Increased sensitivity to inhibition of hematopoiesis by TGF-β has been hypothesized to be a mechanism of hematopoietic failure in Fanconi Anemia (FA). To determine whether abrogation of TGF-β signaling rescued biologic parameters of hematopoiesis, we derived a novel DKO mouse strain (SMAD3^-/- (129/Sv) Fancd2^-/- (C57BL/6)) by breeding double heterozygote mice. The DKO mice were small at birth, but achieved normalized growth and development by six - eight weeks. Hematopoiesis in long-term bone marrow cultures derived from DKO mice, ceased generating day 14 CFU-GEMM hematopoietic progenitors by 18 weeks, similar to that observed with Fancd2^-/- marrow cultures. In contrast, cultures from SMAD3^-/- (129/Sv) demonstrated continuous hematopoiesis for over 30 weeks confirming results with marrow from SMAD3^-/- (C57BL/6J) mice (Epperly, et al., Experimental Hematology, 33:353-362, 2005), and significantly longer than that in control 129/Sv X C57BL/6J F1 mouse long-term marrow cultures. Bone marrow stromal cell lines derived from long-term marrow cultures were tested for the diagnostic criterion of FA, increased sensitivity to DNA cross-linking agent, Mitomycin-C. Cells were incubated in Mitomycin-C at concentrations ranging from 0-20 ng/ml for four days and then plated in 4 well tissue culture plates, incubated for 7 days at 37^o C in a CO2 incubator, stained with crystal violet, and colonies of greater than 50 cells counted. Similar to Fancd2^-/- marrow stromal cells, DKO marrow stromal cells showed increased sensitivity to Mito-C, while marrow stromal cell lines from wild type or SMAD3^-/- mouse marrow cultures were relatively resistant (p = 0.0086 comparing Fancd2-/- to control 129/Sv X C57BL/6J F1 cells, p = 0.0156 comparing DKO with control 129/Sv X C57BL/6J F1 cells, and p = 0.4676 comparing Smad3-/- cells to control 129/Sv X C57BL/6J F1 cells). Fresh bone marrow from DKO mice, similar to that from SMAD3^-/- mice, demonstrated resistance to inhibition of hematopoietic cell colonies in vitro by increasing concentrations of TGF-β. Therefore, abrogation of TGF-β signaling in DKO cells did not alter the sensitivity of Fancd2-/- marrow stromal cells to Mitomycin-C.

Supported by research grant NIAID/NIH, U19A168021.