Consistent Upregulation of Oxidative Stress and DNA Damage Amongst Dyskeratosis Congenita Genotypes: Ameliorative Effects of Antioxidants
Authors: Pereboeva Larisa , Westin Erik , Hubbard Meredith , Goldman Frederick D 
Published: October 2019

Abstract
Introduction:
Dyskeratosis congenita (DC) is an inherited multisystem disorder characterized by the classical clinical triad of leukoplakia, skin dyspigmentation and nail dystrophy with concomitant premature aging, cancer predisposition, and bone marrow failure. Conventional treatments for marrow failure, including transplantation, have been poorly tolerated for DC patients. Several mutations within telomeres/telomerase-associated genes have been attributed to DC, resulting in shortened telomeres. Growing evidence suggests that shortened telomeres are linked to DNA damage responses and subsequent elevation of reactive oxygen species (ROS). We have previously reported increased intracellular ROS and induction of DDR protein expression in TERC-deficient cells. This study was undertaken to confirm these findings in other DC genotypes (TERT, TINF2, DKC1) and provide additional evidence supporting a relationship among telomere shortening, DDR and dysregulation in oxidative pathways.
Methods:
Somatic cells from DC subjects with TERC (5 patients), TERT (4), DKC1 (1) and TINF2 (1) mutations and age-matched controls were used in these studies. Cell types included lymphocytes and fibroblasts from skin and lung. DC genotypes were confirmed by DNA sequencing, and all DC cells displayed shortened telomeres. T lymphocytes were expanded in vitro using CD3/CD28 beads. Ionizing radiation was used to induce DNA damage. Cell proliferation was assessed on a Nexelom automatic counter. Western blotting was used to measure basal and radiation-induced expression of DNA damage response (DDR) proteins, including total p53 and its activated form (serine 15 phosphorylated; p53S15), ATM, BRCA2 and phosphorylated H2AX (gH2AX). Levels of oxidative stress were determined by FACS using the cell-permeable fluorogenic probe DCFH and dihydroethedium (DHE) detecting reactive oxygen species (ROS). Antioxidant culture conditions, such as N acetyl cysteine (NAC) or low oxygen, were used in vitro to modulate levels of oxidative stress in control and radiated cells.
Results:
We investigated ROS levels and several parameters indicative of DNA damage in DC lymphocytes and fibroblasts with mutations in TERC, TERT, DKC1 or TINF2. In cells from all DC genotypes a consistent increase in p53 expression and ROS levels was noted. Basal expression of p53 was increased by at least 50% in DC lymphocytes compared to controls. Additionally, basal ROS levels from TERC and TINF2 mutated lymphocytes were significantly increased compared to controls. While the degree of radiation-induced DDR protein expression was similar in DC compared to control cells, ROS remained significantly higher in DC-irradiated lymphocytes. Lung fibroblasts from a DKC1 patient demonstrated increased ROS and reduced proliferative capacity compared to controls. Importantly, NAC decreased DDR in DC lymphocytes while a low oxygen environment markedly improved the proliferative capacity of both DC lymphocytes and lung fibroblasts.
Conclusions:
DC is an important human disease model for studying the effects of telomere shortening on pathways involved in cell senescence and aging. Our findings indicate elevated basal and irradiation-induced levels of p53 expression and ROS in DC cells regardless of the underlying mutation. This suggests a role for telomerase deficiency and/or shortened telomeres in regulating oxidative and DNA damage response pathways. Furthermore, reducing ROS may serve as an adjunctive therapeutic approach to limit toxicity of bone marrow transplant regimens. Clinical trials are needed to evaluate the impact of antioxidant therapies in this disease.

Disclosures
No relevant conflicts of interest to declare.