[show abstract][hide abstract] ABSTRACT: Telomeres are essential for genomic integrity, but little is known about their regulation in the normal human mammary gland. We now demonstrate that a phenotypically defined cell population enriched in luminal progenitors (LPs) is characterized by unusually short telomeres independently of donor age. Furthermore, we find that multiple DNA damage response proteins colocalize with telomeres in >95% of LPs but in <5% of basal cells. Paradoxically, 25% of LPs are still capable of exhibiting robust clonogenic activity in vitro. This may be partially explained by the elevated telomerase activity that was also seen only in LPs. Interestingly, this potential telomere salvage mechanism declines with age. Our findings thus reveal marked differences in the telomere biology of different subsets of primitive normal human mammary cells. The chronically dysfunctional telomeres unique to LPs have potentially important implications for normal mammary tissue homeostasis as well as the development of certain breast cancers.
[show abstract][hide abstract] ABSTRACT: Telomerase activity is readily detectable in extracts from human hematopoietic stem and progenitor cells, but appears unable to maintain telomere length with proliferation in vitro and with age in vivo. We performed a detailed study of the telomere length by flow FISH analysis in leukocytes from 835 healthy individuals and 60 individuals with reduced telomerase activity. Healthy individuals showed a broad range in average telomere length in granulocytes and lymphocytes at any given age. The average telomere length declined with age at a rate that differed between age-specific breakpoints and between cell types. Gender differences between leukocyte telomere lengths were observed for all cell subsets studied; interestingly, this trend could already be detected at birth. Heterozygous carriers for mutations in either the telomerase reverse transcriptase (hTERT) or the telomerase RNA template (hTERC) gene displayed striking and comparable telomere length deficits. Further, non-carrier relatives of such heterozygous individuals had somewhat shorter leukocyte telomere lengths than expected; this difference was most profound for granulocytes. Failure to maintain telomere homeostasis as a result of partial telomerase deficiency is thought to trigger cell senescence or cell death, eventually causing tissue failure syndromes. Our data are consistent with these statements and suggest that the likelihood of similar processes occurring in normal individuals increases with age. Our work highlights the essential role of telomerase in the hematopoietic system and supports the notion that telomerase levels in hematopoietic cells, while limiting and unable to prevent overall telomere shortening, are nevertheless crucial to maintain telomere homeostasis with age.
[show abstract][hide abstract] ABSTRACT: Studies of telomeres and telomere biology often critically rely on the detection of telomeric DNA and measurements of the length of telomere repeats in either single cells or populations of cells. Several methods are available that provide this type of information and it is often not clear what method is most appropriate to address a specific research question. The major variables that need to be considered are the material that is or can be made available and the accuracy of measurements that is required. The goal of this review is to provide a comprehensive summary of the most commonly used methods and discuss the advantages and disadvantages of each. Methods that start with genomic DNA include telomere restriction fragment (TRF) length analysis, PCR amplification of telomere repeats relative to a single copy gene by Q-PCR or MMQPCR and single telomere length analysis (STELA), a PCR-based approach that accurately measures the full spectrum of telomere lengths from individual chromosomes. A different set of methods relies on fluorescent in situ hybridization (FISH) to detect telomere repeats in individual cells or chromosomes. By including essential calibration steps and appropriate controls these methods can be used to measure telomere repeat length or content in chromosomes and cells. Such methods include quantitative FISH (Q-FISH) and flow FISH which are based on digital microscopy and flow cytometry, respectively. Here the basic principles of various telomere length measurement methods are described and their strengths and weaknesses are highlighted. Some recent developments in telomere length analysis are also discussed. The information in this review should facilitate the selection of the most suitable method to address specific research question about telomeres in either model organisms or human subjects.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 06/2011; 730(1-2):59-67. · 3.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dyskeratosis congenita (DC) is an inherited bone marrow (BM) failure syndrome associated with mutations in telomerase genes and the acquisition of shortened telomeres in blood cells. To investigate the basis of the compromised hematopoiesis seen in DC, we analyzed cells from granulocyte colony-stimulating factor mobilized peripheral blood (mPB) collections from 5 members of a family with autosomal dominant DC with a hTERC mutation. Premobilization BM samples were hypocellular, and percentages of CD34(+) cells in marrow and mPB collections were significantly below values for age-matched controls in 4 DC subjects. Directly clonogenic cells, although present at normal frequencies within the CD34(+) subset, were therefore absolutely decreased. In contrast, even the frequency of long-term culture-initiating cells within the CD34(+) DC mPB cells was decreased, and the telomere lengths of these cells were also markedly reduced. Nevertheless, the different lineages of mature cells were produced in normal numbers in vitro. These results suggest that marrow failure in DC is caused by a reduction in the ability of hematopoietic stem cells to sustain their numbers due to telomere impairment rather than a qualitative defect in their commitment to specific lineages or in the ability of their lineage-restricted progeny to execute normal differentiation programs.
[show abstract][hide abstract] ABSTRACT: Telomeres play a central role in cell fate and aging by adjusting the cellular response to stress and growth stimulation on the basis of previous cell divisions and DNA damage. At least a few hundred nucleotides of telomere repeats must "cap" each chromosome end to avoid activation of DNA repair pathways. Repair of critically short or "uncapped" telomeres by telomerase or recombination is limited in most somatic cells and apoptosis or cellular senescence is triggered when too many "uncapped" telomeres accumulate. The chance of the latter increases as the average telomere length decreases. The average telomere length is set and maintained in cells of the germline which typically express high levels of telomerase. In somatic cells, telomere length is very heterogeneous but typically declines with age, posing a barrier to tumor growth but also contributing to loss of cells with age. Loss of (stem) cells via telomere attrition provides strong selection for abnormal and malignant cells, a process facilitated by the genome instability and aneuploidy triggered by dysfunctional telomeres. The crucial role of telomeres in cell turnover and aging is highlighted by patients with 50% of normal telomerase levels resulting from a mutation in one of the telomerase genes. Short telomeres in such patients are implicated in a variety of disorders including dyskeratosis congenita, aplastic anemia, pulmonary fibrosis, and cancer. Here the role of telomeres and telomerase in human aging and aging-associated diseases is reviewed.
[show abstract][hide abstract] ABSTRACT: Little is known about the behavior of hematopoietic stem cells (HSCs) in primates because direct observations and competitive-repopulation assays are not feasible. Therefore, we used 2 different and independent experimental strategies, the tracking of transgene expression after retroviral-mediated gene transfer (N = 11 baboons; N = 7 rhesus macaques) and quantitation of the average telomere length of granulocytes (N = 132 baboons; N = 14 macaques), together with stochastic methods, to study HSC kinetics in vivo. The average replication rate for baboon HSCs is once per 36 weeks according to gene-marking analyses and once per 23 weeks according to telomere-shortening analyses. Comparable results were derived from the macaque data. These rates are substantially slower than the average replication rates previously reported for HSCs in mice (once per 2.5 weeks) and cats (once per 8.3 weeks). Because baboons and macaques live for 25 to 45 years, much longer than mice ( approximately 2 years) and cats (12-18 years), we can compute that HSCs undergo a relatively constant number ( approximately 80-200) of lifetime replications. Thus, our data suggest that the self-renewal capacity of mammalian stem cells in vivo is defined and evolutionarily conserved.