Telomeres, stem cells, and hematology

Abstract

Telomeres are highly dynamic structures that adjust the cellular response to stress and growth stimulation based on previous cell divisions. This critical function is accomplished by progressive telomere shortening and DNA damage responses activated by chromosome ends without sufficient telomere repeats. Repair of critically short 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 germ line that typically express high levels of telomerase. In somatic cells, the telomere length 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 cells in which malignant progression is facilitated by genome instability resulting from uncapped telomeres. The critical role of telomeres in cell proliferation and aging is illustrated in patients with 50% of normal telomerase levels resulting from a mutation in one of the telomerase genes. Here, the role of telomeres and telomerase in human biology is reviewed from a personal historical perspective.

Figure 1

Developmental changes and telomere loss in hematopoietic cells. (A) Ontogeny-related differences in the production of CD34⁺ cells in culture. (Reproduced from Lansdorp et al with permission.) Candidate stem cells with a CD34⁺CD45RA^lowCD71^low phenotype were purified from fetal liver (wk12), umbilical cord blood, and adult bone marrow and 10⁴ sorted cells were cultured in serum-free culture medium supplemented with IL-6, IL-3, steel factor, and erythropoietin as described. At the indicated time interval, the total number of nucleated cells (light blue squares) and CD34⁺ cells (dark blue squares) present in the cultures was calculated from cell counts and the percentage of viable CD34⁺ cells measured by flow cytometry. All CD34⁺ cells from bone marrow cultures and fractions of the CD34⁺ cells from cord blood and fetal liver cultures were sorted and used for continuation of the cultures. (B) Loss of telomeric DNA in human hematopoietic cells upon proliferation in vivo and in vitro (reproduced from Vaziri et al with permission from the National Academy of Sciences of the United States). DNA samples from total nucleated cells from 2 different donors for each tissue before and after culture at increasing time points were subjected to terminal restriction fragment (TRF) size Southern blot analysis as described elsewhere. Cultures were initiated with highly enriched stem cells as described in panel A. (C) Quantitative analysis of the TRF data shown in panel B. The first time point (blue square with circle) represents the TRF value in nucleated cells from each tissue before purification. The mean and standard error of 2 independent TRF measurements (different gels) for each DNA sample purified from total nucleated cells at each time point are shown. The total number of cells was used to calculate population doublings. The loss of telomeric DNA in these cultures varied between 19 and 54 base pairs per population doubling. For details see Vaziri et al.

Figure 2

Fluorescence in situ hybridization to detect telomere repeats. Quantitative in situ hybridization (Q-FISH) using Cy3-labeled (CCCTAA)₃ peptide nucleic acid probes on metaphase chromosomes from a human lymphocyte (A) and an embryonic fibroblast from a late-generation telomerase RNA KO mouse (B). Note that the fluorescence intensity is very similar for both sister chromatids at individual chromosome ends but very heterogeneous between ends. The arrows in panel B point to pseudo metacentric chromosomes resulting from the fusion of 2 acrocentric mouse chromosomes. Note the lack of telomere repeats at the junction of the 2 fused chromosomes.

Figure 3

Telomere length measurements using flow FISH. (A) Nonlinear decline in telomere length with age. The average telomere length in granulocytes and lymphocytes from 392 healthy donors was calculated from the median telomere fluorescence and median autofluorescence relative to internal control cells (bovine thymocytes). Note that the telomere length at any given age is highly variable, that the most rapid drop in telomere length occurs early in life, and that the rate of telomere attrition in lymphocytes exceeds that in granulocytes. (B) Longitudinal studies of telomere length in newborn baboons point to a high turnover of hematopoietic stem cells in the first year of life. Note that the 2 animals differ markedly in average telomere length and that the rate of telomere attrition drops markedly after approximately 1 to 2 years in both animals. In humans, with a longer lifespan, this drop is expected to occur before the fourth year of life in line with previous cross-sectional observations. (C,D) Telomere length in individuals with mutations in telomere genes. The telomere length in cells from healthy individuals (A) was used to plot the telomere length distribution in the normal population using a best-fit approach (red, green, and blue curves representing expected telomere length for the indicated proportion of healthy individuals). The telomere length in lymphocytes (C) and granulocytes (D) from patients with known mutations in telomerase genes measured in the context of several studies^–,, are shown. Each symbol represents an individual patient diagnosed with clinical symptoms associated with a mutation in dyskerin (DKC1, black circles), hTERT (red circles), and hTERC (black squares). Some individuals are carriers of a TERC mutation but have no clinical symptoms (blue square). The majority of individuals that carry mutations in telomerase genes display critically short telomeres, nearly all of them below the tenth percentile of the normal distribution and a majority of these below the first percentile (typically for both cell subsets shown). Note that individuals with early onset of disease (in the first 3 decades of life) show the most striking difference between observed and expected telomere length.