Characterization and rescue of telomeric abnormalities in ICF syndrome type I fibroblasts.

Mutations in the human DNA methyltransferase 3B (DNMT3B) gene lead to ICF (immunodeficiency, centromeric region instability, and facial anomalies) syndrome type I. We have previously described a telomere-related phenotype in cells from these patients, involving severe hypomethylation of subtelomeric regions, abnormally short telomeres and high levels of telomeric-repeat-containing RNA (TERRA). Here we demonstrate that ICF-patient fibroblasts carry abnormally short telomeres at a low population doubling (PD) and enter senescence prematurely. Accordingly, we attempted to rescue the senescence phenotype by ectopic expression of human telomerase, which led to elongated telomeres with hypomethylated subtelomeres. The senescence phenotype was overcome under these conditions, thus dissociating subtelomeric-DNA hypomethylation per se from the senescence phenotype. In addition, we examined whether the subtelomeric methylation could be restored by expression of a normal copy of full length DNMT3B1 in ICF fibroblasts. Ectopic expression of DNMT3B1 failed to rescue the abnormal hypomethylation at subtelomeres. However, partial rescue of subtelomeric-hypomethylation was achieved by co-expression of DNMT3B1 together with DNA methyltransferase 3-like (DNMT3L), encoding a protein that functions as a stimulator of DNMT3A and DNMT3B. DNMT3B1 and DNMT3L are predominantly expressed during early embryonic development, suggesting that de novo subtelomeric DNA methylation during crucial stages of human embryonic development may be necessary for setting and maintaining normal telomere length.

Quantitative digital in situ senescence-associated ß-galactosidase assay.

BACKGROUND: Cellular senescence plays important roles in the aging process of complex organisms, in tumor suppression and in response to stress. Several markers can be used to identify senescent cells, of which the most widely used is the senescence-associated ß-galactosidase (SABG) activity. The main advantage of SABG activity over other markers is the simplicity of the detection assay and the capacity to identify in situ a senescent cell in a heterogeneous cell population. Several approaches have been introduced to render the SABG assay quantitative. However none of these approaches to date has proven particularly amenable to quantitative analysis of SABG activity in situ. Furthermore the role of cellular senescence (CS) in vivo remains unclear mainly due to the ambiguity of current cellular markers in identifying CS of individual cells in tissues. RESULTS: In the current study we applied a digital image analysis technique to the staining generated using the original SABG assay, and demonstrate that this analysis is highly reproducible and sensitive to subtle differences in staining intensities resulting from diverse cellular senescence pathways in culture. We have further validated our method on mouse kidney samples with and without diabetes mellitus, and show that a more accurate quantitative SABG activity with a wider range of values can be achieved at a pH lower than that used in the conventional SABG assay. CONCLUSIONS: We conclude that quantitative in situ SABG assay, is feasible and reproducible and that the pH at which the reaction is performed should be tailored and chosen, depending on the research question and experimental system of interest.

Telomere elongation followed by telomere length reduction, in leukocytes from divers exposed to intense oxidative stress--implications for tissue and organismal aging.

Many cross-sectional studies have tried to assess the in vivo effect of oxidative stress on organismal aging in general and on telomere length dynamics specifically. Here we followed telomere length dynamics over a 12-month interval, in divers exposed to intense hyperbaric oxygen in comparison with an age-matched control group. Both groups were exposed to extreme physical activity, as well. Among the divers following the oxidative stress, significant telomere elongation was observed in granulocytes and naïve T cells, but not in memory T cells and B cells. Telomere length in granulocytes was mildly elongated in the control group as well, a finding that may relate to the extreme physical activity to which they were exposed. While telomere elongation in naïve T cells may be attributed to telomerase activation, we suggest that in granulocytes the elongation results from undifferentiated hematopoietic cells carrying longer telomeres that repopulate the peripheral hematopoietic compartment. This event might be accompanied by enhanced cell division within the repopulating pool. Since the aging of mammalian tissues can be attributed in part to the reduction in the replicative potential of self renewing cells, enhanced cell turnover under conditions of hyperbaric oxidative stress might be directly relevant to tissue and organismal aging.