SummaryDyskeratosis congenita (DC), an inherited bone marrow failure syndrome, is caused by defects in telomerase. Somatic cells from DC patients have shortened telomeres and clinical symptoms are most pronounced in organs with a high cell turnover, including those involved in hematopoiesis and skin function. We previously identified an autosomal dominant (AD) form of DC that is caused by mutations in the telomerase RNA component (TER). In this study, we evaluated whether retroviral expression of TER and/or telomerase reverse transcriptase (TERT), the catalytic component of telomerase, could extend telomere length and rescue AD DC cells from a phenotype characteristic of early senescence. Exogenous TER expression, without TERT, could not activate telomerase in AD DC skin fibroblasts. Transduction of TERT alone, however, provided AD DC cells with sufficient telomerase activity to extend average telomere length and proliferative capacity. Interestingly, we found that expression of TER and TERT together resulted in extension of lifespan and higher levels of telomerase and longer telomeres than expression of TERT alone in both AD DC and normal cells. Our results provide evidence that AD DC cells can be rescued from defects in telomere maintenance and proliferation, and that coexpression of TERT and TER together provides a more efficient means to elongate telomeres than expression of TERT alone. Similar strategies may be useful for ameliorating the detrimental effects of telomere shortening in AD DC and other diseases associated with telomerase or telomere defects.

Mutations of the Janus family kinase JAK3 gene cause severe combined immunodeficiency (SCID). JAK3 deficiency in humans is characterized by the absence of circulating T cells and natural killer (NK) cells with normal numbers of poorly functioning B cells (T(-)B(+)NK(-)). Using SCID patient-specific induced pluripotent stem cells (iPSCs) and a T cell in vitro differentiation system, we demonstrate a complete block in early T cell development of JAK3-deficient cells. Correction of the JAK3 mutation by CRISPR/Cas9-enhanced gene targeting restores normal T cell development, including the production of mature T cell populations with a broad T cell receptor (TCR) repertoire. Whole-genome sequencing of corrected cells demonstrates no CRISPR/Cas9 off-target modifications. These studies describe an approach for the study of human lymphopoiesis and provide a foundation for gene correction therapy in humans with immunodeficiencies.

Telomere attrition is a natural process that occurs due to inadequate telomere maintenance. Once at a critically short threshold, telomeres signal growth arrest, leading to senescence. Telomeres can be elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Mutations in genes for telomere binding proteins or components of telomerase give rise to the premature aging disorder dyskeratosis congenita (DC), which is characterized by extremely short telomeres and an aging phenotype. The current study demonstrates that DC cells signal a DNA damage response through p53 and its downstream mediator, p21 WAF=CIP , which is accompanied by an elevation in steady-state levels of superoxide and percent glutathione disulfide, both indicators of oxidative stress. Poor proliferation of DC cells can be partially overcome by reducing O 2 tension from 21% to 4%. Further, restoring telomerase activity or inhibiting p53 or p21 WAF=CIP significantly mitigated growth inhibition as well as caused a significant decrease in steady-state levels of superoxide. Our results support a model in which telomerase insufficiency in DC leads to p21 WAF=CIP signaling, via p53, to cause increased steady-state levels of superoxide, metabolic oxidative stress, and senescence. Antioxid. Redox Signal. 14, 985-997.

Dyskeratosis congenita (DC) is an inherited multisystem disorder of premature aging, cancer predisposition, and bone marrow failure caused by selective exhaustion of highly proliferative cell pools. DC patients also have a poor tolerance to chemo/radiotherapy and bone marrow transplantation. Although critically shortened telomeres and defective telomere maintenance contribute to DC pathology, other mechanisms likely exist. We investigate the link between telomere dysfunction and oxidative and DNA damage response pathways and assess the effects of antioxidants. In vitro studies employed T lymphocytes from DC subjects with a hTERC mutation and age-matched controls. Cells were treated with cytotoxic agents, including Paclitaxel, Etoposide, or ionizing radiation. Apoptosis and reactive oxygen species (ROS) were assessed by flow cytometry, and Western blotting was used to measure expression of DNA damage response (DDR) proteins, including total p53, p53S15, and p21WAF. N-acetyl-cysteine (NAC), an antioxidant, was used to modulate cell growth and ROS. In stimulated culture, DC lymphocytes displayed a stressed phenotype, characterized by elevated levels of ROS, DDR and apoptotic markers as well as a proliferative defect that was more pronounced after exposure to cytotoxic agents. NAC partially ameliorated the growth disadvantage of DC cells and decreased radiation-induced apoptosis and oxidative stress. These findings suggest that oxidative stress may play a role in the pathogenesis of DC and that pharmacologic intervention to correct this pro-oxidant imbalance may prove useful in the clinical setting, potentially alleviating untoward toxicities associated with current cytotoxic treatments.

The interaction between electromagnetic radiation and the C 60 molecule has been analysed by an evaluation of the low-energy part ( 40 eV) of the linear response spectrum of buckminsterfullerene, C 60 . Single-particle wavefunctions, determined from local-density ground-state calculations, were used for evaluation of dipole matrix elements which, combined in a sum-over-states approach, yielded wavelength-dependent free-response microscopic linear polarizabilities. Screening of the external electromagnetic field through the induced charge distribution was determined by applying external static electric fields in separate self-consistent field calculations and by evaluation of the induced dipole moment. The polarizability calculated in the static limit was used to determine an effective screening parameter for evaluation of the dynamic response. The inclusion of screening was found to give an optical spectrum in rather good agreement with the corresponding experimentally determined spectrum of C 60 , in the gas phase or solution, by absorption measurements, EELS or photoionization techniques. Furthermore, by the use of Lorentz local field factors, we find a similar agreement for the dielectric function (ω) determined from C 60 ellipsometry and EELS measurements. In the above cases, the shapes of the spectra are essentially reproduced except for a uniform shift of about 0.5 eV to the low-energy side in the region 10 eV and about 4 eV in the opposite direction in the region above 10 eV.