Reactivation of Telomerase in Late Generation Tert−/− Mice Results in Reversal of Anemia

Abstract 1258

We previously reported that telomere shortening in a late generation telomerase K/O mouse model results in significantly reduced RBC and erythroblast (CD71+Ter119+) numbers in the peripheral blood and bone marrow, respectively. We also observed significant decreases in erythroid progenitor (Lin-cKit+Sca1-CD34-CD16/CD32-) and HSC (Lin-cKit+Sca1+ CD34-CD150+) numbers in G5 Tert−/− mice. We now report the mechanism underlying the specific defect in the erythroid lineage in these mice. First, there is a higher number of CD34-KLS CD150lo and CD150neg cells in the mutant mice relative to age matched G0 +/− mice. Methyl cellulose colony formation assay showed that CD34-KLS CD150hi cells give rise to a greater number of CFU-GEMM colonies while CD34-KLS CD150lo and CD34-KLS CD150neg cells both had a reduction in the numbers of GEMM colonies, suggesting that the loss of erythroid differentiation is associated with loss of CD150hi expression within the CD34-KLS population. Thus, a significant reduction in CD150hi HSC (p value < 0.001) and an increase in CD150lo (p value < 0.001) and CD150neg (p value < 0.04) HSC in G5 Tert−/− mice underlies the decreased number of erythroblasts in these mice. Secondly, we found that the reduction in telomere length as measured by quantitative PCR is greater in sorted erythroblasts (CD71+Ter119+) than in similarly purified myeloid progenitors (Gr1+Mac1+) in G5 Tert −/− mice. This finding correlates with the presence of increased DNA damage and apoptosis in erythroid progenitors and erythroblasts as compared to myeloid progenitors. In order to establish a cause and effect relationship between telomere shortening and defective erythropoiesis, we reactivated telomerase activity using a tamoxifen-inducible Cre recombinase to excise a Lox-Stop-Lox cassette located in intron 2 of telomerase gene in G5 Tert−/− mice. Reactivation of telomerase activity resulted in increased CD150hi HSC, erythroid progenitors and erythroblasts numbers in the bone marrow of these mice 3 months after tamoxifen treatment. In addition, RBC numbers and Hb levels in the peripheral blood improved in the treated mice, whereas neither improved in control G5 Tert−/− mice treated with sunflower oil. These data show for the first time a direct link between the loss of telomerase function, telomere shortening and a selective defect in erythropoiesis. These results, in conjunction with single-cell “mass cytometry” experiments underway to define the intracellular signaling pathways that are affected in the HSC and erythroid precursors of G5 Tert−/− mice, demonstrate the lineage-specific effects of telomere shortening on erythropoiesis and help to elucidate the relationship between shortened telomeres and the anemia of bone marrow failure syndromes.