DNA Damage Accumulation, Accelerated Hematopoietic Stem Cell Aging, and Partial Reversal by Antioxidant Treatment in a Mouse Model of Dyskeratosis Congenita.

Abstract

Abstract 498X-linked dyskeratosis congenita (DC) caused by mutations in DKC1, encoding the protein dyskerin, is the most common form of DC, a severe inherited bone marrow failure (BMF) syndrome associated with a predisposition to malignancy. Dyskerin is a component of small nucleolar ribonucleoprotein particles(snoRNPs) that modify specific residues in nascent ribosomal RNA(rRNA) molecules and also forms part of the telomerase complex responsible for synthesizing the telomere repeats at the ends of chromosomes. Strong evidence suggests that compromised telomerase function is the major cause of DC but defects in ribosome biogenesis may contribute to the disease. Excessive telomere shortening resulting in premature cellular senescence is thought to be the primary cause of bone marrow failure in dyskeratosis congenita. Our previous data showed that, in mice, cells expressing a Dkc1 mutation (Dkc1Δ15) had a telomerase dependent but telomere length independent growth defect. Here we show that the growth rate of Δ15 MEF cells was lower when cultured at both ambient oxygen (21%) and low (3%) oxygen. In 21% oxygen both Δ15 and WT cells stopped growing and entered senescence after 8-10 population doublings, with the Δ15 cells growing more slowly than the WT cells. In 3% oxygen Δ15 cells grew more slowly and entered senescence earlier than WT cells. Further investigations reveal that both γ-H2AX foci number and reactive oxygen species (ROS) levels in Δ15 cells were significantly higher than in WT cells with increased passage number even when cultured in low oxygen. Increased levels of γ-H2AX and p53 in Dkc1Δ15 mice, particularly in older mice, were also detected in liver, spleen and bone marrow. To study the effect of the mutation on stem cell function during aging, we carried out competitive repopulation experiments using the CD45.1/CD45.2 congenic system. Irradiated mice were injected with a 1:1 mixture of Dkc1Δ15 and Dkc1+ bone marrow from old (77-88w) or young (10w) animals. Old Dkc1Δ15cells are less able to compete with age matched WT cells in primary recipients, making up only 20% of cells after 12 weeks compared with 40% for the young cells. Moreover, serial transplantation results show that, in secondary recipients, BM cells from old Dkc1Δ15 mice were not detectable while Dkc1Δ15 cells from young mice still comprise 10-30% of the bone marrow after 12 weeks. These results strongly indicate the Dkc1Δ15 mutation causes decay of stem cell function with age. Because of the association with ROS we asked whether treatment with an antioxidant could rescue the growth disadvantage of Δ15 cells. We grew primary MEF cells from Dkc1Δ15/+ female mice in the presence or absence of 100 M N-acetyl cysteine (NAC), a clinically approved antioxidant. These cultures consist in early passages of 50% cells expressing WT and 50% expressing Δ15 dyskerin, reflecting random X-chromosome inactivation, Without NAC the WT cells almost completely outgrew the Δ15 cells after 11 population doublings but in the presence of NAC the Δ15 cells are still clearly present after 15 population doublings, suggesting that NAC at least partially rescues the growth disadvantage of dyskerin mutant cells. More impressively, the growth disadvantage of the Δ15 cells is also rescued in vivo in Dkc1Δ15/+ female mice given the NAC (1mg/ml) in their drinking water. Although the precise mechanism will be the subject of further investigation, these results point to a functional link between increased oxidative stress, defective telomere maintenance and stem cell aging in the pathogenesis of BMF in dyskeratosis congenita. Disclosures:Bessler:Alexion: Membership on an entity's Board of Directors or advisory committees.