Body mass index and leukocyte telomere length dynamics among older adults: Results from the ESTHER cohort.

OBJECTIVE: Telomere length (TL) has been proposed as a biomarker of ageing, which might be used to identify individuals at higher risk of age-related diseases. Obesity is a well-known risk factor for several diseases. This study aims to analyse the associations of BMI with TL and the rate of TL change in older adults. METHODS: Leukocyte TL (LTL) was measured by quantitative PCR in blood samples of 3600 older adults aged 50-75 years obtained at the baseline examination of a population-based cohort study in Germany. For longitudinal analyses, measurements were repeated in blood samples obtained at 8-year follow-up from 1000 participants. Multivariate linear regression models were used to estimate associations of BMI with LTL and changes in LTL over time. RESULTS: LTL was inversely associated with age (r = -0.090, p < 0.0001). BMI and LTL associations varied according to age (p for interaction = 0.021). BMI was significantly inversely associated with LTL in those younger than 60 years (-6 basepairs per 1 kg/m(2) difference in BMI). In particular, weight gain during adulthood was inversely associated with LTL in a dose-response manner in this age group, with those having gained ≥ 30 kg having significantly shorter telomeres (-209 basepairs) than those who maintained their weight. No clear patterns were observed between any of BMI-related variables and the rate of LTL change. CONCLUSIONS: Our cross-sectional analysis supports suggestions that weight gain during adulthood and obesity may contribute to shorter telomere length below 60 years of age, but this relationship could not be shown longitudinally.

Smoking habits and leukocyte telomere length dynamics among older adults: Results from the ESTHER cohort.

BACKGROUND & AIMS: Leukocyte telomere length (LTL) shortens with age and short LTL has been associated with increased mortality and increased risk for some age-related outcomes. This study aims to analyse the associations of smoking habits with LTL and rate of LTL change per year in older adults. METHODS: LTL was measured by quantitative PCR at baseline in 3600 older adults, who were enrolled in a population-based cohort study in Germany. For longitudinal analyses, measurements were repeated in blood samples obtained at 8-year follow-up from 1000 participants. Terminal Restriction Fragment analysis was additionally performed in a sub-sample to obtain absolute LTL in base pairs. Multivariate linear regression models were used to estimate associations of smoking habits with baseline LTL and changes in LTL over time. RESULTS: LTL was inversely associated with age (r=-0.090, p<0.0001). Women had longer LTL than men (p<0.0001). Smoking was inversely associated with LTL. On average, current smokers had 73 base pairs (BP) shorter LTL compared to never smokers. Smoking intensity and pack-years of smoking were also inversely associated with LTL, and a positive association was observed with years since smoking cessation. Slower LTL attrition rates were observed in ever smokers over 8years of follow-up. CONCLUSIONS: Our cross-sectional analysis supports suggestions that smoking might contribute to shortening of LTL but this relationship could not be shown longitudinally. The overall rather small effect sizes observed for smoking-related variables suggest that LTL reflects smoking-related health hazards only to a very limited extent.

Wnt-3a-activated human fibroblasts promote human keratinocyte proliferation and matrix destruction.

Aberrant Wnt regulation, detectable by nuclear translocation of beta-catenin, is a hallmark of many cancers including skin squamous cell carcinomas (SCCs). By analyzing primary human skin SCCs, we demonstrate that nuclear beta-catenin is not restricted to SCC cells but also detected in stromal fibroblasts, suggesting an important role for aberrant Wnt regulation also in the tumor microenvironment. When human keratinocytes and fibroblasts were treated with Wnt-3a, fibroblasts proved to be more responsive. Accordingly, Wnt-3a did not alter HaCaT cell functions in a cell-autonomous manner. However, when organotypic cultures (OTCs) were treated with Wnt-3a, HaCaT keratinocytes responded with increased proliferation. As nuclear beta-catenin was induced only in the fibroblasts, this argued for a Wnt-dependent, paracrine keratinocyte stimulation. Global gene expression analysis of Wnt-3a-stimulated fibroblasts identified genes encoding interleukin-8 (IL-8) and C-C motif chemokine 2 (CCL-2) as well as matrix metalloproteinase-1 (MMP-1) as Wnt-3a targets. In agreement, we show that IL-8 and CCL-2 were secreted in high amounts by Wnt-3a-stimulated fibroblasts also in OTCs. The functional role of IL-8 and CCL-2 as keratinocyte growth regulators was confirmed by directly stimulating HaCaT cell proliferation in conventional cultures. Most important, neutralizing antibodies against IL-8 and CCL-2 abolished the Wnt-dependent HaCaT cell hyperproliferation in OTCs. Additionally, MMP-1 was expressed in high amounts in Wnt-3a-stimulated OTCs and degraded the stromal matrix. Thus, our data show that Wnt-3a stimulates fibroblasts to secrete both keratinocyte proliferation-inducing cytokines and stroma-degrading metalloproteinases, thereby providing evidence for a novel Wnt deregulation in the tumor-stroma directly contributing to skin cancer progression.

Frailty and telomere length: cross-sectional analysis in 3537 older adults from the ESTHER cohort.

Both telomere length and frailty were observed to be associated with aging. Whether and to what extent telomere length is related to frailty is essentially unknown. In this cross-sectional analysis of baseline data of 3537 community-dwelling adults aged 50 to 75 years of a large German cohort study, we assessed the hypothesis that shorter telomere length might be a biological marker for frailty. Using whole blood DNA we examined mean telomere repeat copy to single gene copy number (T/S ratio) using quantitative PCR. Construction of a frailty index (FI) was based on a deficit accumulation approach, which quantifies frailty as ratio of the deficits present divided by the total number of deficits considered. Mean FI was determined according to age by tertiles of T/S ratio. Furthermore, we used correlation analyses stratified for gender and age groups to examine the association of the T/S ratio with frailty. Mean FI value was similar across tertiles of the T/S ratio (0.24±0.14, 0.24±0.14 and 0.23±0.14, respectively (p=0.09)), and FI and the T/S ratio were uncorrelated in gender- and age-specific analyses. In conclusion, T/S ratio and frailty were unrelated in this large sample of older adults. T/S ratio may therefore not be a meaningful biological marker for frailty.

Tissue context-activated telomerase in human epidermis correlates with little age-dependent telomere loss.

Telomerase- and telomere length regulation in normal human tissues is still poorly understood. We show here that telomerase is expressed in the epidermis in situ independent of age but was repressed upon the passaging of keratinocytes in monolayer culture. However, when keratinocytes were grown in organotypic cultures (OTCs), telomerase was re-established, indicating that telomerase activity is not merely proliferation-associated but is regulated in a tissue context-dependent manner in human keratinocytes. While not inducible by growth factors, treatment with the histone deacetylation inhibitor FK228 restored telomerase activity in keratinocytes grown in monolayer cultures. Accordingly, CHIP analyses demonstrated an acetylated, active hTERT promoter in the epidermis in situ and in the epidermis of OTCs but a deacetylated, silenced hTERT promoter with subsequent propagation in monolayer culture suggesting that histone acetylation is part of the regulatory program to guarantee hTERT expression/telomerase activity in the epidermis. In agreement with the loss of telomerase activity, telomeres shortened during continuous propagation in monolayer culture by an average of approximately 70 base pairs (bp) per population doubling (pd). However, telomere erosion varied strongly between different keratinocyte strains and even between individual cells within the same culture, thereby arguing against a defined rate of telomere loss per replication cycle. In the epidermis in situ, as determined from early-passage keratinocytes and tissue sections from different age donors, we calculated a telomere loss of only approximately 25 bp per year. Since we determined the same rate for the non-regenerating melanocytes and dermal fibroblasts, our data suggest that in human epidermis telomerase is a protective mechanism against excessive telomere loss during the life-long regeneration.

Proteasome-mediated degradation antagonizes critical levels of the apoptosis-inducing C1D protein.

The C1D gene is expressed in a broad spectrum of mammalian cells and tissues but its product induces apoptotic cell death when exceeding a critical level. Critical levels are achieved in a fraction of cells by transient transfection with EGFP-tagged C1D expression constructs. However, transfected cells expressing sub-critical levels of C1D(EGFP) escape apoptotic cell death by activation of a proteasome-mediated rescue mechanism. Inhibition of the proteasome-dependent degradation of the C1D(EGFP) protein results in a parallel increase of the intracellular C1D level and in the fraction of apoptotic cells.

Cytotoxic cyplasin of the sea hare, Aaplysia punctata, cDNA cloning, and expression of bioactive recombinants in insect cells.

A 56-kDa protein isolated from the mucus of the European sea hare Aplysia punctata shows a prefer ential toxicity to autonomously growing transformed mammalian cells. Cell death induced by this protein differs from both apoptosis and necrosis. The cytotoxic effects are irreversible and become apparent at nanomolar concentrations in a cell type-dependent manner. In contrast, injection of micromolar concentrations into mice is tolerated without apparent negative consequences. Microsequencing of the 56-kDa protein released a peptide sequence whose corresponding nucleotide sequence was used as probe to screen A. punctata RNA-based cDNA and to select cDNA clones encoding polypeptides comprising the target peptide. Two closely related cDNA were detected. The cDNA encoding a polypeptide 558 aa in length was considered to reflect a bonafide clone encoding the cytotoxic protein. Its protein-coding section was recloned in vectors suitable for expression in Escherichia coli, in mammalian cells, and in insect cells, respectively. The E. coli-expressed polypeptide was biologically inactive. Transfected mammalian cells expressed a cytotoxic factor and died thereof as if treated with the genuine cytotoxic protein. In contrast, transfected insect cells, which proved to be much less sensitive when treated with the genuine protein, expressed the cytotoxic factor and continued to proliferate, allowing to establish stable insect cell lines expressing sufficient amounts of the cytotoxic factor for further characterization.