Induced pluripotent stem cells as a model for telomeric abnormalities in ICF type I syndrome.

Human telomeric regions are packaged as constitutive heterochromatin, characterized by extensive subtelomeric DNA methylation and specific histone modifications. ICF (immunodeficiency, centromeric instability, facial anomalies) type I patients carry mutations in DNA methyltransferase 3B (DNMT3B) that methylates de novo repetitive sequences during early embryonic development. ICF type I patient fibroblasts display hypomethylated subtelomeres, abnormally short telomeres and premature senescence. In order to study the molecular mechanism by which the failure to de novo methylate subtelomeres results in accelerated telomere shortening, we generated induced pluripotent stem cells (iPSCs) from 3 ICF type I patients. Telomeres were elongated in ICF-iPSCs during reprogramming, and the senescence phenotype was abolished despite sustained subtelomeric hypomethylation and high TERRA levels. Fibroblast-like cells (FLs) isolated from differentiated ICF-iPSCs maintained abnormally high TERRA levels, and telomeres in these cells shortened at an accelerated rate, leading to early senescence, thus recapitulating the telomeric phenotype of the parental fibroblasts. These findings demonstrate that the abnormal telomere phenotype associated with subtelomeric hypomethylation is overridden in cells expressing telomerase, therefore excluding telomerase inhibition by TERRA as a central mechanism responsible for telomere shortening in ICF syndrome. The data in the current study lend support to the use of ICF-iPSCs for modeling of phenotypic and molecular defects in ICF syndrome and for unraveling the mechanism whereby subtelomeric hypomethylation is linked to accelerated telomeric loss in this syndrome.

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.

Hypomethylation of subtelomeric regions in ICF syndrome is associated with abnormally short telomeres and enhanced transcription from telomeric regions.

Telomeres and adjacent subtelomeric regions are packaged as heterochromatin in many organisms. The heterochromatic features include DNA methylation, histones H3-Lys9 (Lysine 9) and H4-Lys20 (Lysine 20) methylation and heterochromatin protein1 alpha binding. Subtelomeric DNA is hypomethylated in human sperm and ova, and these regions are subjected to de novo methylation during development. In mice this activity is carried out by DNA methyltransferase 3b (Dnmt3b). Mutations in DNMT3B in humans lead to the autosomal-recessive ICF (immunodeficiency, centromeric region instability, facial anomalies) syndrome. Here we show that, in addition to several satellite and non-satellite repeats, the subtelomeric regions in lymphoblastoid and fibroblast cells of ICF patients are also hypomethylated to similar levels as in sperm. Furthermore, the telomeres are abnormally short in both the telomerase-positive and -negative cells, and many chromosome ends lack detectable telomere fluorescence in situ hybridization signals from either one or both sister-chromatids. In contrast to Dnmt3a/b(-/-) mouse embryonic stem cells, increased telomere sister-chromatid exchange was not observed in ICF cells. Hypomethylation of subtelomeric regions was associated in the ICF cells with advanced telomere replication timing and elevated levels of transcripts emanating from telomeric regions, known as TERRA (telomeric-repeat-containing RNA) or TelRNA. The current findings provide a mechanistic explanation for the abnormal telomeric phenotype observed in ICF syndrome and highlights the link between TERRA/TelRNA and structural telomeric integrity.

Energetic efficiency in trained and sedentary rats after exposure to normobaric and hyperbaric oxygen.

INTRODUCTION: Contradictory results have been obtained regarding the beneficial effect of hyperbaric oxygen (HBO) on exercise performance. The purpose of this study was to investigate the effect of different combinations of pressure and time in hyperoxia on the energetic efficiency of trained and sedentary rats. METHODS: At the end of the training period, rats were exposed to one of three protocols: 1) 100% normobaric oxygen for 24 h; 2) HBO at 2 ATA for 4 h; 3) HBO at 2.5 ATA for 6 h. After the hyperoxic exposures, V(O2max) was evaluated and compared with preexposure values. RESULTS: The slope of the linear section of the oxygen consumption-velocity curve in the trained rats was significantly steeper after exposure to either 100% normobaric oxygen for 24 h or HBO at 2 ATA for 4 h than before the exposure. The opposite was found for the sedentary rats. After exposure to HBO at 2.5 ATA for 6 h, the slope of the oxygen consumption-velocity curve in the trained rats did not differ from the pre-exposure slope. However, the highest velocity these rats reached was lower than their maximum velocity before this exposure. In the sedentary rats, the slope of the oxygen consumption-velocity curve was found to be steeper after the 2.5 ATA exposure compared with the preexposure slope. CONCLUSIONS: Our results suggest that exposure to 100% normobaric oxygen for 24 h and HBO at 2 ATA for 4 h induces a reduction in the energetic efficiency of trained rats, but improves energetic efficiency in sedentary rats.

The Therapeutic Potential of AN-7, a Novel Histone Deacetylase Inhibitor, for Treatment of Mycosis Fungoides/Sezary Syndrome Alone or with Doxorubicin.

The 2 histone deacetylase inhibitors (HDACIs) approved for the treatment of cutaneous T-cell lymphoma (CTCL) including mycosis fungoides/sezary syndrome (MF/SS), suberoylanilide hydroxamic acid (SAHA) and romidepsin, are associated with low rates of overall response and high rates of adverse effects. Data regarding combination treatments with HDACIs is sparse. Butyroyloxymethyl diethylphosphate (AN-7) is a novel HDACI, which was found to have selective anticancer activity in several cell lines and animal models. The aim of this study was to compare the anticancer effects of AN-7 and SAHA, either alone or combined with doxorubicin, on MF/SS cell lines and peripheral blood lymphocytes (PBL) from patients with Sezary syndrome (SPBL). MyLa cells, Hut78 cells, SPBL, and PBL from healthy normal individuals (NPBL) were exposed to the test drugs, and the findings were analyzed by a viability assay, an apoptosis assay, and Western blot. AN-7 was more selectively toxic to MyLa cells, Hut78 cells, and SPBL (relative to NPBL) than SAHA and also acted more rapidly. Both drugs induced apoptosis in MF/SS cell lines, SAHA had a greater effect on MyLa cell line, while AN-7 induced greater apoptosis in SPBL; both caused an accumulation of acetylated histone H3, but AN-7 was associated with earlier kinetics; and both caused a downregulation of the HDAC1 protein in MF/SS cell lines. AN-7 acted synergistically with doxorubicin in both MF/SS cell lines and SPBL, and antagonistically with doxorubicin in NPBL. By contrast, SAHA acted antagonistically with doxorubicin on MF/SS cell lines, SPBL, and NPBL, leaving <50% viable cells. In conclusion, AN-7 holds promise as a therapeutic agent in MF/SS and has several advantages over SAHA. Our data provide a rationale for combining AN-7, but not SAHA, with doxorubicin to induce the cell death in MF/SS.

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.

Reprogramming of telomeric regions during the generation of human induced pluripotent stem cells and subsequent differentiation into fibroblast-like derivatives.

Human induced pluripotent stem (hiPS) cells provide therapeutic promises, as well as a potent in vitro model for studying biological processes which take place during human embryonic development and subsequent differentiation in normal and disease states. The epigenetic characteristics of iPS cells are reprogrammed to the embryonic state at which they acquire pluripotency. In addition, telomeres in hiPS cell must elongate sufficiently to provide the necessary replicative potential. Recent studies have demonstrated that the epigenetic characteristics of telomeric and subtelomeric regions are pivotal in regulating telomere length. Here we study telomere length, subtelomeric DNA methylation and telomeric-repeat-containing RNA (TERRA) expression in several hiPS cell clones derived from normal neonatal foreskin fibroblasts. We find that telomeres lengthen significantly in hiPS cells in comparison to the parental fibroblast source, and progressively shorten after differentiation back into fibroblast-like cells, concomitantly with telomerase activation and down-regulation, respectively. Subtelomeres in hiPS cells were found to be generally hypermethylated in comparison to the parental source. However bisulfite analysis revealed that at several subtelomeres examined, methylation levels differed between hiPS clones and that both de novo methylation and demethylation processes occurred during telomere reprogramming. Notably, although subtelomeres were in general very highly methylated, TERRA levels were elevated in hiPS cells, albeit to different degrees in the various clones. TERRA elevation may reflect enhanced stability or impaired degradation in hiPS cells, and/or alternatively, increased transcription from the hypomethylated subtelomeres. We suggest that TERRA may play a role in regulation of appropriate telomere function and length in hiPS cells.

Functional characterization of SAMD9, a protein deficient in normophosphatemic familial tumoral calcinosis.

Dystrophic cutaneous calcinosis is associated with disorders as common as autoimmune diseases and cancer. To get insight into the pathogenesis of this poorly understood process, we studied the function of SAMD9, a protein of unknown function, recently shown to be deficient in a hereditary form of dystrophic calcification in the skin, known as normophosphatemic familial tumoral calcinosis (NFTC). Consistent with the fact that in NFTC severe inflammatory manifestations always precede cutaneous calcinosis, we found out that SAMD9 is tightly regulated by interferon-γ (IFN-γ). In addition, the SAMD9 promoter was also found to respond strongly to IFN-γ in a luciferase reporter assay. Of interest, we identified a critical 30-bp fragment upstream to the SAMD9 transcription initiation site responsible for driving most of the gene expression. Bioinformatic analysis suggested that SAMD9 function involves interaction with additional protein(s). Using the Ras recruitment system assay and confirmatory immunoprecipitation, we demonstrated that SAMD9 interacts with RGL2. To study the biological importance of this interaction, we assessed the effect of RNA interference-mediated downregulation of this pair of proteins in various cell lines. We found out that downregulation of any of the two protein partners caused increased expression of EGR1, a transcription factor with a known role in the regulation of tissue calcification, inflammation, and cell migration. Supporting the physiological relevance of these data, EGR1 levels were also upregulated in a fibroblast cell line derived from an NFTC patient. In conclusion, our data indicate that SAMD9, an IFN-γ-responsive protein, interacts with RGL2 to diminish the expression of EGR1, a protein of direct relevance to the pathogenesis of ectopic calcification and inflammation.

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.