Leukocyte Telomere Length in Children With Congenital Adrenal Hyperplasia.

CONTEXT: Exposure to chronic stress and hypercortisolism is associated with decreased leukocyte telomere length (LTL), a marker for biological aging and cardiovascular disease. Children with congenital adrenal hyperplasia (CAH) are treated with glucocorticoids. OBJECTIVE: To investigate LTL in children with CAH. METHODS: In this prospective observational cohort study, conducted at 4 academic pediatric endocrinology outpatient clinics, children with genetically confirmed CAH were assessed at 2 follow-up visits (mean 4.1 ± 0.7 months apart). At each visit, LTL was determined by quantitative real-time PCR. All subjects underwent detailed clinical and endocrinologic evaluation and were classified as undertreated, optimally treated, or overtreated, accordingly. The influence of clinical factors on LTL was investigated using linear mixed models adjusted for age, sex, and BMI-z. RESULTS: We studied 76 patients, of whom 31 (41%) were girls, 63 (83%) had classic CAH, 67 (88%) received hydrocortisone, and 8 (11%) prednisolone. Median age at first visit was 12.0 years (IQR, 6.3-15.1), and median BMI-z was 0.51 (IQR, -0.12 to 1.43). LTL was shorter in patients with classic vs nonclassic CAH (-0.29, P = 0.012), in overtreated than in optimally treated patients (-0.07, P = 0.002), and patients receiving prednisolone compared with hydrocortisone (-0.34, P < 0.001). LTL was not associated with undertreatment or daily hydrocortisone-equivalent dose (P > 0.05). CONCLUSION: LTL is shorter in patients with classic than nonclassic CAH, and in those who are overtreated with hydrocortisone or treated with long-acting glucocorticoids. These findings may be attributed to chronic exposure to supraphysiologic glucocorticoid concentrations and indicate that LTL may be used as a biomarker for monitoring glucocorticoid treatment.

Desmin deficiency affects the microenvironment of the cardiac side population and Sca1+ stem cell population of the adult heart and impairs their cardiomyogenic commitment.

The heart's limited regenerative capacity raises the need for novel stem cell-based therapeutic approaches for cardiac regeneration. However, the use of stem cells is restrictive due to poor determination of their properties and the factors that regulate them. Here, we investigated the role of desmin, the major muscle-specific intermediate filament protein, in the characteristics and differentiation capacity of cardiac side population (CSP) and Sca1+ stem cells of adult mice. We found that desmin deficiency affects the microenvironment of the cells and leads to increased numbers of CSP but not Sca1+ cells; CSP subpopulation composition is altered, the expression of the senescence marker p16INK4a in Sca1+ cells is increased, and early cardiomyogenic commitment is impaired. Specifically, we found that mRNA levels of the cardiac transcription factors Mef2c and Nkx2.5 were significantly reduced in des-/- CSP and Sca1+ cells, while differentiation of CSP and Sca1+ cells demonstrated that in the absence of desmin, the levels of Nkx2.5, Mef2c, Tnnt2, Hey2, and Myh6 mRNA are differentially affected. Thus, desmin deficiency restricts the regenerative potential of CSP and Sca1+ cells, both directly and indirectly through their microenvironment.

Association between Telomere Length and Pediatric Obesity: A Systematic Review.

OBJECTIVE: Telomere length (TL) is a robust marker of biological aging, and increased telomere attrition is noted in adults with obesity. The primary objective of this systematic review was to summarize current knowledge on the effects of childhood obesity in TL. The secondary objective was to assess the effect of weight management interventions in TL. METHODS: The following databases were searched: PubMed, Scopus, Web of Science and Heal-link.gr from inception to September 2021. The search was performed using the following combinations of terms: "telomer*" [All Fields] AND ("length" [All Fields] OR "lengths" [All Fields]) AND "obes*" [All Fields] AND ("child*" [All Fields] OR "adolescen*" [All Fields]). RESULTS: A total of 16 original articles were included in this systematic review. Eleven of them were cross-sectional and five were lifestyle interventions. CONCLUSIONS: There was a tendency towards a negative association between childhood obesity and TL. Life-style interventions in children have been associated with increased TL peripherally, indicating a possible association of the redistribution of younger cells in the periphery with the favorable effect of these interventions. Further prospective studies with larger sample sizes that employ other markers of cell aging would potentially elucidate this important mechanistic relation.

A Comprehensive, Multidisciplinary, Personalized, Lifestyle Intervention Program Is Associated with Increased Leukocyte Telomere Length in Children and Adolescents with Overweight and Obesity.

Leucocyte telomere length (LTL) is a robust marker of biological aging and is associated with obesity and cardiometabolic risk factors in childhood and adolescence. We investigated the effect of a structured, comprehensive, multidisciplinary, personalized, lifestyle intervention program of healthy diet and physical exercise on LTL in 508 children and adolescents (239 males, 269 females; 282 prepubertal, 226 pubertal), aged 10.14 ± 0.13 years. Participants were classified as obese (n = 267, 52.6%), overweight (n = 174, 34.2%), or of normal BMI (n = 67, 13.2%) according to the International Obesity Task Force (IOTF) cutoff points and were studied prospectively for one year. We demonstrated that LTL increased significantly after 1 year of the lifestyle interventions, irrespective of gender, pubertal status, or body mass index (BMI). Waist circumference was the best negative predictor of LTL at initial assessment. The implementation of the lifestyle interventions also resulted in a significant improvement in clinical (BMI, BMI z-score and waist to height ratio) and body composition indices of obesity, inflammatory markers, hepatic enzymes, glycated hemoglobin (HbA1C), quantitative insulin sensitivity check index (QUICKI), and lipid profile in all participants. These findings indicate that the increased LTL may be associated with a more favorable metabolic profile and decreased morbidity later in life.

Karyotypic Flexibility of the Complex Cancer Genome and the Role of Polyploidization in Maintenance of Structural Integrity of Cancer Chromosomes.

Ongoing chromosomal instability in neoplasia (CIN) generates intratumor genomic heterogeneity and limits the efficiency of oncotherapeutics. Neoplastic human cells utilizing the alternative lengthening of telomeres (ALT)-pathway, display extensive structural and numerical CIN. To unravel patterns of genome evolution driven by oncogene-replication stress, telomere dysfunction, or genotoxic therapeutic interventions, we examined by comparative genomic hybridization five karyotypically-diverse outcomes of the ALT osteosarcoma cell line U2-OS. These results demonstrate a high tendency of the complex cancer genome to perpetuate specific genomic imbalances despite the karyotypic evolution, indicating an ongoing process of genome dosage maintenance. Molecular karyotyping in four ALT human cell lines showed that mitotic cells with low levels of random structural CIN display frequent evidence of whole genome doubling (WGD), suggesting that WGD may protect clonal chromosome aberrations from hypermutation. We tested this longstanding hypothesis in ALT cells exposed to gamma irradiation or to inducible DNA replication stress under overexpression of p21. Single-cell cytogenomic analyses revealed that although polyploidization promotes genomic heterogeneity, it also protects the complex cancer genome and hence confers genotoxic therapy resistance by generating identical extra copies of driver chromosomal aberrations, which can be spared in the process of tumor evolution if they undergo unstable or unfit rearrangements.

ApoE isoforms and carboxyl-terminal-truncated apoE4 forms affect neuronal BACE1 levels and Aß production independently of their cholesterol efflux capacity.

The ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) initiates the production of amyloid-ß peptide (Aß), which is central to the pathogenesis of Alzheimer's disease (AD). Changes in brain cholesterol homeostasis have been suggested to affect Aß metabolism. Cholesterol homeostasis is maintained in the brain by apolipoprotein E (apoE). The apoE4 isoform constitutes the major risk factor for AD. Here, we investigated the effect of apoE forms on Aß generation and on BACE1 levels. We also examined the potential involvement in these processes of cholesterol transporters ABCG1 and ABCG4 or the lipoprotein receptor SR-BI, which are implicated in cholesterol efflux to apoE. It was found that reconstituted lipoprotein-associated apoE isoforms promoted the increase of Aß production and oligomerization and of BACE1 levels in human neuroblastoma SK-N-SH cells, with an apoE4 ≥ apoE3 > apoE2 potency rank order. Progressive carboxyl-terminal apoE4 deletions between residues 230-299 decreased the protein's ability to increase BACE1, while further truncations up to residue 166 prevented apoE4 from increasing BACE1 and Aß levels in SK-N-SH and primary mouse neuronal cells. ABCG1, but not ABCG4 or SR-BI, moderately increased Aß production and BACE1 levels in SK-N-SH cells. All apoE forms affected Aß production/oligomerization and BACE1 levels in a pattern that did not follow that of their capacity to promote ABCG1, ABCG4 or SR-BI-mediated cholesterol efflux. Overall, our data indicate that apoE-containing lipoprotein particles can have a direct effect on BACE1 levels and Aß secretion and possibly contribute to AD pathogenetic processes, independently of their capacity to promote cholesterol efflux.

Alternative lengthening of telomeres: recurrent cytogenetic aberrations and chromosome stability under extreme telomere dysfunction.

Human tumors using the alternative lengthening of telomeres (ALT) exert high rates of telomere dysfunction. Numerical chromosomal aberrations are very frequent, and structural rearrangements are widely scattered among the genome. This challenging context allows the study of telomere dysfunction-driven chromosomal instability in neoplasia (CIN) in a massive scale. We used molecular cytogenetics to achieve detailed karyotyping in 10 human ALT neoplastic cell lines. We identified 518 clonal recombinant chromosomes affected by 649 structural rearrangements. While all human chromosomes were involved in random or clonal, terminal, or pericentromeric rearrangements and were capable to undergo telomere healing at broken ends, a differential recombinatorial propensity of specific genomic regions was noted. We show that ALT cells undergo epigenetic modifications rendering polycentric chromosomes functionally monocentric, and because of increased terminal recombinogenicity, they generate clonal recombinant chromosomes with interstitial telomeric repeats. Losses of chromosomes 13, X, and 22, gains of 2, 3, 5, and 20, and translocation/deletion events involving several common chromosomal fragile sites (CFSs) were recurrent. Long-term reconstitution of telomerase activity in ALT cells reduced significantly the rates of random ongoing telomeric and pericentromeric CIN. However, the contribution of CFS in overall CIN remained unaffected, suggesting that in ALT cells whole-genome replication stress is not suppressed by telomerase activation. Our results provide novel insights into ALT-driven CIN, unveiling in parallel specific genomic sites that may harbor genes critical for ALT cancerous cell growth.

The roles of telomerase in the generation of polyploidy during neoplastic cell growth.

Polyploidy contributes to extensive intratumor genomic heterogeneity that characterizes advanced malignancies and is thought to limit the efficiency of current cancer therapies. It has been shown that telomere deprotection in p53-deficient mouse embryonic fibroblasts leads to high rates of polyploidization. We now show that tumor genome evolution through whole-genome duplication occurs in ∼15% of the karyotyped human neoplasms and correlates with disease progression. In a panel of human cancer and transformed cell lines representing the two known types of genomic instability (chromosomal and microsatellite), as well as the two known pathways of telomere maintenance in cancer (telomerase activity and alternative lengthening of telomeres), telomere dysfunction-driven polyploidization occurred independently of the mutational status of p53. Depending on the preexisting context of telomere maintenance, telomerase activity and its major components, human telomerase reverse transcriptase (hTERT) and human telomerase RNA component (hTERC), exert both reverse transcriptase-related (canonical) and noncanonical functions to affect tumor genome evolution through suppression or induction of polyploidization. These new findings provide a more complete mechanistic understanding of cancer progression that may, in the future, lead to novel therapeutic interventions.

Human UPF1 interacts with TPP1 and telomerase and sustains telomere leading-strand replication.

Eukaryotic up-frameshift 1 (UPF1) is a nucleic acid-dependent ATPase and 5'-to-3' helicase, best characterized for its roles in cytoplasmic RNA quality control. We previously demonstrated that human UPF1 binds to telomeres in vivo and its depletion leads to telomere instability. Here, we show that UPF1 is present at telomeres at least during S and G2/M phases and that UPF1 association with telomeres is stimulated by the phosphoinositide 3-kinase (PI3K)-related protein kinase ataxia telangiectasia mutated and Rad3-related (ATR) and by telomere elongation. UPF1 physically interacts with the telomeric factor TPP1 and with telomerase. Akin to UPF1 binding to telomeres, this latter interaction is mediated by ATR. Moreover, the ATPase activity of UPF1 is required to prevent the telomeric defects observed upon UPF1 depletion, and these defects stem predominantly from inefficient telomere leading-strand replication. Our results portray a scenario where UPF1 orchestrates crucial aspects of telomere biology, including telomere replication and telomere length homeostasis.

A non-canonical function of zebrafish telomerase reverse transcriptase is required for developmental hematopoiesis.

Although it is clear that telomerase expression is crucial for the maintenance of telomere homeostasis, there is increasing evidence that the TERT protein can have physiological roles that are independent of this central function. To further examine the role of telomerase during vertebrate development, the zebrafish telomerase reverse transcriptase (zTERT) was functionally characterized. Upon zTERT knockdown, zebrafish embryos show reduced telomerase activity and are viable, but develop pancytopenia resulting from aberrant hematopoiesis. The blood cell counts in TERT-depleted zebrafish embryos are markedly decreased and hematopoietic cell differentiation is impaired, whereas other somatic lineages remain morphologically unaffected. Although both primitive and definitive hematopoiesis is disrupted by zTERT knockdown, the telomere lengths are not significantly altered throughout early development. Induced p53 deficiency, as well as overexpression of the anti-apoptotic proteins Bcl-2 and E1B-19K, significantly relieves the decreased blood cells numbers caused by zTERT knockdown, but not the impaired blood cell differentiation. Surprisingly, only the reverse transcriptase motifs of zTERT are crucial, but the telomerase RNA-binding domain of zTERT is not required, for rescuing complete hematopoiesis. This is therefore the first demonstration of a non-canonical catalytic activity of TERT, which is different from "authentic" telomerase activity, is required for during vertebrate hematopoiesis. On the other hand, zTERT deficiency induced a defect in hematopoiesis through a potent and specific effect on the gene expression of key regulators in the absence of telomere dysfunction. These results suggest that TERT non-canonically functions in hematopoietic cell differentiation and survival in vertebrates, independently of its role in telomere homeostasis. The data also provide insights into a non-canonical pathway by which TERT functions to modulate specification of hematopoietic stem/progenitor cells during vertebrate development. (276 words).

Pericentromeric instability and spontaneous emergence of human neoacrocentric and minute chromosomes in the alternative pathway of telomere lengthening.

In the alternative pathway of telomere lengthening (ALT), neoplastic cell growth is prolonged by telomere recombination. We show that ALT is unexpectedly characterized by high rates of ongoing pericentromeric chromosomal instability. Combined with telomeric recombination, ALT pericentromeric instability generates neoacrocentric chromosomes. In the present studies, we describe a subgroup of ALT neoacrocentric minute chromosomes, composed of DNA entities two to five times smaller in size than human chromosome 21. The frequencies of ALT minute chromosomes were increased by gamma-irradiation and suppressed by telomerase. Continuous growth after telomerase inhibition/depletion was followed by increased rates of telomeric sister chromatid recombination and the emergence of minute chromosomes. We show that ALT minute chromosomes were derived from true centromeric fissions and/or chromosomal breakage/fusion/bridge cycles. They exhibit a two-chromatid structure, carry genomic DNA, centromeric and telomeric repeats, and display regular mitotic functionality. These observations are important in understanding the global genomic instability that characterizes most human advanced malignancies.