Abstract
Abstract 2359
Introduction:
Dyskeratosis congenita (DC) is an inherited multisystem disorder consisting of premature aging, cancer predisposition, bone marrow failure and the characteristic triad of mucosal leukoplakia, skin dyspigmentation and nail dystrophy. Symptomology associated with DC arises as a consequence of mutations within genes associated with telomeres and telomerase activity manifested by critically shortened telomeres in affected cells. We have previously reported a growth disadvantage and increased intracellular oxidative stress in cultured somatic cells obtained from patients with DC. We hypothesize that telomere maintenance is closely linked to dysregulation in oxidative pathways and consequent DNA damage. Our objective was to discern whether pharmacologic intervention to alleviate oxidative stress imparts a protective effect in DC cells.
Methods:
T lymphocytes from both DC subjects with hTERC mutations and age-matched controls were cultured and expanded in vitro using CD3/CD28 beads. DNA damage to cells was induced using paclitaxel, etoposide, or ionizing radiation during log-phase of cell growth. Cellular proliferation and apoptosis were monitored by cell counting and flow cytometry (FACS) using Annexin V antibody and propidium iodide. 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), p21WAF, and phosphorylated H2AX (gH2AX). Level of oxidative stress was determined by FACS using the cell-permeable fluorogenic probe DCFH and dihydroethedium (DHE) detecting reactive oxygen species (ROS). Anti-oxidants, including vitamin E and N acetyl cysteine (NAC), were used in vitro to modulate levels of oxidative stress in control and radiated cells.
Results:
Comparison of growth curves demonstrated a significant decrease in proliferation of T cells obtained from DC patients versus control T cells. This growth disadvantage was more pronounced following cell exposure to radiation, paclitaxel, and etoposide. To explain these differences we investigated several parameters indicative of DNA damage. DC lymphocytes had higher basal levels of apoptosis, while radiation resulted in comparable levels of apoptosis in both DC and control cultures. Similarly, DDR markers p53 and p53S15, but not p21 and g-H2AX, were basally expressed at higher levels in DC lymphocytes while radiation, in a dose-dependent manner, upregulated expression of p53, p53S15, p21 and g-H2AX in both DC and control lymphocytes. Consistent with DDR data, elevated basal levels of ROS were found in short term DC cultures. Additionally, in a dose dependent manner, the anti-oxidant NAC partially ameliorated the growth disadvantage of DC cells. Importantly, NAC also decreased radiation-induced apoptosis and oxidative stress in DC cells. Studies are ongoing to characterize the modulation of DDR markers in NAC-treated cells.
Conclusions:
DC is an important disease model for studying the effects of telomere shortening on cellular proliferation and other molecular pathways involved in cell senescence and aging. Our findings of elevated basal levels of apoptosis, DDR proteins and oxidative stress in DC lymphocytes, as well as increased sensitivity of DC cells to cytotoxic agents suggests a role of telomerase and/or telomere length in regulating oxidative and DNA damage response pathways. This data also validates the clinical finding of DC patients' intolerance to myeloablative therapy. Finally a pharmacologic approach to reduce oxidative stress may alleviate some of the untoward toxicities associated with current cytotoxic treatments in DC. Clinical trials testing various anti-oxidant therapies are currently under design.
Disclosures:
No relevant conflicts of interest to declare.
In vitro toxic evaluation of two gliptins and their main impurities of synthesis
