Telomeric proteins have an essential role in the regulation of the length of the telomeric DNA tract and in protection against end-to-end chromosome fusion. Telomere organization and how individual proteins are involved in different telomere functions in living cells is largely unknown. By using green fluorescent protein tagging and photobleaching, we investigated in vivo interactions of human telomeric DNA-binding proteins with telomeric DNA. Our results show that telomeric proteins interact with telomeres in a complex dynamic fashion: TRF2, which has a dual role in chromosome end protection and telomere length homeostasis, resides at telomeres in two distinct pools. One fraction (ϳ73%) has binding dynamics similar to TRF1 (residence time of ϳ44 s). Interestingly, the other fraction of TRF2 binds with similar dynamics as the putative end-protecting factor hPOT1 (residence time of ϳ11 min). Our data support a dynamic model of telomeres in which chromosome end-protection and telomere length homeostasis are governed by differential binding of telomeric proteins to telomeric DNA.
Telomeres, nucleoprotein complexes at chromosome ends, protect chromosomes against end-to-end fusion. Previous in vitro studies in human fibroblast models indicated that telomere dysfunction results in chromosome instability. Loss of telomere function can result either from critical shortening of telomeric DNA or from loss of distinct telomere-capping proteins. It is less clear whether telomere dysfunction has an important role in human cancer development in vivo. Acute myeloid leukemia (AML) is a good model to study mechanisms that generate chromosome instability in human cancer development because distinct groups of AML are characterized either by aberrations that theoretically could result from telomere dysfunction (terminal deletions, gains/losses of chromosome parts, nonreciprocal translocations), or aberrations that are unlikely to result from telomere dysfunction (e.g., reciprocal translocations or inversions). Here we demonstrate that AML with multiple chromosome aberrations that theoretically could result from telomere dysfunction is invariably characterized by critically short telomeres. Short telomeres in this group are not associated with low telomerase activity or decreased expression of essential telomeric capping proteins TRF2 and POT1. In contrast, telomerase activity levels are significantly higher in AML with short telomeres. Notably, short telomeres in the presence of high telomerase may relate to significantly higher expression of TRF1, a negative regulator of telomere length. Our observations suggest that, consistent with previous in vitro fibroblast models, age-related critical telomere shortening may have a role in generating chromosome instability in human AML development.
SummaryAge is an important factor in the development and spread of breast cancer. Stromal cells also contribute to breast cancer growth and metastasis through the production of extracellular matrix (ECM) modifiers such as urokinase type plasminogen activator (uPA), its receptor (uPAR), its inhibitors (PAI-1 and PAI-2), matrix metalloproteinases (MMPs), and growth factors, including the fibroblast and insulin-like growth factors (FGF’s and IGF’s). In the present study we have investigated whether breast fibroblasts aged in vitro through passage in culture display altered levels of the plasminogen activator system and growth factors that are known to modulate that system.With real-time RT-PCR we found that during passage human breast fibroblasts, whether derived from the tumour burden or from matched adjacent normal breast tissue, exhibited a consistent increase in PAI-1 and FGF-1 and a decrease in MMP-2 mRNA expression. In addition, in 5 out of 7 fibroblast strains we observed an induction of uPA expression in combination with a reduced IGF-1 expression. Interestingly, while during aging MMP-2 protein increased in all tumour-derived fibroblast strains, these protein levels were reduced in all normal-tissue-derived fibroblasts. No other clear-cut age-dependent alterations were found in the all-together 25 factors investigated. We furthermore demonstrate in one tumour-derived fibroblast strain that the increases in uPA and PAI-1 mRNA and MMP-2 protein production are inversely related to the telomere length. Artificially increasing telomere length in this fibroblast strain by expressing human telomerase reverse transcriptase (hTERT) prevented senescence and resulted in late passage cultures with early passage uPA, PAI-1 and MMP-2 levels.Our results show that aging accompanied by telomere loss induces PAI-1 and FGF-1 mRNA expression in all breast fibroblast strains, increases uPA and decreases IGF-1 mRNA expression in a subset, and increases MMP-2 protein expression only in tumour-derived breast fibroblasts. These age-induced levels of PAI-1, FGF-1, uPA and MMP-2 in stromal breast fibroblast could contribute to breast cancer progression.
Telomeres, the ends of linear chromosomes, have a critical role in protection against chromosome end-to-end fusion. Telomeres shorten in every cell division due to the end replication problem. Telomerase is a reverse transcriptase that adds telomeric DNA repeats to the ultimate chromosome end. In vitro models of long-term fibroblast cultures have identified two sequential mortality stages, senescence (M1) and crisis (M2). Senescence can be bypassed by loss of p53 or Rb function, whereas escape from crisis can only be achieved by activating a telomere maintenance mechanism, mostly telomerase reactivation. Cells that bypass senescence (M1) did not reactivate telomerase, resulting in further telomere shortening to a critical telomere length upon reaching crisis (M2). In these models, critical telomere shortening induces extensive chromosome instability, most likely via chromosome end-to-end fusions. Dicentric chromosomes lead to anaphase breakage-fusion-bridges resulting in multiple chromosomal aberrations. To investigate whether similar mechanisms may be involved in the development of genetic instability in human cancer, we studied telomere length and expression of critical telomeric proteins (TRF2 and POT1) in acute myeloid leukemia (AML) patients. AML is a good model for these studies since distinct subgroups of AML are characterized by either exchanges along chromosome arms (translocation or inversion), or by a complex karyotype with multiple chromosome aberrations. Groups were age-matched. Telomere length was studied in metaphase arrested leukemic cells using quantitative fluorescence in situ hybridization (Q-FISH) using a telomere-specific probe. Subsequently, metaphase spreads were hybridized with a leukemia-specific probe to confirm leukemic origin of each metaphase. Telomeres were significantly shorter in AML samples with multiple chromosomal abnormalities in comparison to AML samples with a reciprocal translocation/inversion or no abnormalities (mean±SEM=16±1.7 AFU, n=12 versus 29±4.3 AFU, n=18; p=0.015). Interestingly, telomerase activity level is significantly higher in AML samples with multiple chromosomal abnormalities, compared to AML samples with a reciprocal translocation or inversion (mean±SEM=330±95, n=11 versus 70±21, n=13; p=0.02). Expression levels of telomeric proteins TRF2 and POT1 were similar in these AML groups. Our observations suggest that, consistent with previous in vitro models in fibroblasts, critical telomere shortening may have a role in the development of genetic instability in human AML. Critically short telomeres in association with high levels of telomerase activity suggest that AML cells with multiple chromosomal abnormalities have bypassed crisis (M2). The longer telomeres and low levels of telomerase activity in AML cells with a reciprocal translocation or inversion suggest that they originate from an earlier stage, preceding crisis. Consequently, telomere length modulation may have a role in cancer prevention.
Immortal cell growth is considered the hallmark of tumor cells. In contrast, normal cells have a limited proliferative capacity of 40–60 cell divisions, also known as the Hayflick limit. The limited proliferative capacity of normal cells relates to gradual telomere shortening as a consequence of the end-replication problem. Upon critical telomere shortening, cells enter a non-replicative but viable state referred to as replicative senescence. These replicative senescent cells stain blue in a beta-Galactosidase assay and activate DNA double-strand break repair pathways at telomeres (e.g. gamma-H2AX foci). In human fibroblast models, escape from senescence results from loss of p53 and Rb function. Escape is associated with reactivation of telomerase. High levels of telomerase, as observed in germ cells and most tumor cells, allow for immortal cell growth. Recently, we demonstrated low levels of telomerase in AML patients with t(8;21) or inv(16) (Swiggers et al, G.C.C. 2006). Interestingly, levels of telomerase in these AML samples were similar to levels of telomerase in normal bone marrow progenitor cells. We hypothesized that AML without re-activated telomerase may still have intact senescence pathways that limit the proliferative capacity of normal cells. This hypothesis was addressed by studying AML patient samples without telomerase re-activation, i.e., t(8;21), t(15;17) or inv(16) (n=10), and a control group of AML with telomerase re-activation (multiple gains/losses of genetic material, n=8). AML samples werelong-time cultured in vitro in the presence of hematopoietic growth factors (range 3–6 weeks),analyzed in vivo following transplantation in NOD-SCID mice andin patients at time of relapse. Cells with all characteristics of replicative senescence, i.e. enlarged, viable, non-proliferating, blue-coloring in beta-Galactosidase assay, critical short telomeres and gamma-H2AX foci at telomeres, were clearly observed in all AML samples with t(8;21), t(15;17) or inv(16). Gradual telomere shortening was observed in these AML cells in vitro upon long-term culture, in vivo after transplantation in NOD-SCID mice and in vivo in patients at relapse compared to time of diagnosis, indicating that these AML cells do not have an adequate telomere maintenance mechanism. We conclude that AML cells with t(8;21), t(15;17) or inv(16) are characterized by intact pathways that induce replicative senescence. Intact pathways that limit proliferative lifespan may be critical to the high cure rates following chemotherapy treatment of patients with good-risk AML.
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