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Telomeres and telomerase in cell senescence and oncogenesis

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Titel Telomeres and telomerase in cell senescence and oncogenesis
Looptijd 01 / 2007 - onbekend
Status Lopend
URL http://www1.erasmusmc.nl/hematologie/index.php?cId=89
Onderzoeknummer OND1324886
Leverancier gegevens Website EMC

Samenvatting (EN)

Replicative cellular senescence. The proliferative capacity of normal somatic cells is limited. For several decades, this process of replicative cellular senescence has been studied predominantly in fibroblast in vitro models. Primary fibroblasts proliferate in vitro for up to 50 cell divisions, i.e. the Hayflick limit. Then, cells stop dividing and enter a status that is referred to as replicative cellular senescence. Previously, senescence was attributed to the accumulation of random DNA damage. Recently, a critical role of telomeres in replicative senescence has been demonstrated. Modulation of telomeres results in an increased proliferative capacity of primary fibroblasts to >250 cell divisions, i.e. ~>5 fold the normal life span. Telomeres. Telomeres are the physical ends of linear chromosomes. Telomeres are complex protein/DNA structures. In mammals, telomeric DNA consists of a variable number of TTAGGG repeats. Conventional DNA polymerase cannot replicate the 3 end of a linear strand of DNA, i.e. the end replication problem. This inability of DNA polymerase causes gradual shortening of chromosomes in every cell division. Telomeres have two basic functions. First, telomeres are essential for chromosome stability since they protect against end-to-end chromosome fusion and subsequent degradation. Most cancer cells, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cells, are characterized by short telomeres. Short telomeres and specific telomere binding proteins, e.g. TRF-2 in humans, may be critical for chromosome instability in cancer cells. The second function of the telomere is to provide a substrate for elongation of the chromosome end through the enzyme telomerase. Telomerase. The enzyme telomerase consists of a RNA and protein component. The telomerase RNA is complementary to the 3 telomeric DNA sequence and acts as a template for the addition of a single six base repeat to the 3 chromosome end. Consequently, telomerase is a reverse (RNA to DNA) transcriptase. The telomerase catalytic protein component has recently been identified. In agreement with its function, the catalytic protein component of telomerase shows homology to other reverse transcriptases. Telomerase is active in germ line cells and in most cancer cells. It maintains chromosome integrity in immortal cells by compensating for the end replication problem. Hematopoietic and epithelial stem cells express low level of telomerase, i.e. ~1% of the telomerase activity that is observed in most tumor cells. This low level of telomerase activity is not sufficient to maintain telomeres in stem cells following in vitro proliferation. In telomerase deficient mice, loss of function is observed specifically in the reproductive and hematopoietic organs, i.e. the systems that are characterized by a rapid cell turnover. This loss of function is associated with critical telomere shortening. Description of research: general outline. The research addresses mechanisms of aging in the blood cell system with a focus on the role of gradual loss of chromosome ends, i.e. telomeres. We address clinically important questions on the role of cell aging in leukemia formation. In addition, more fundamental questions to elucidate telomere structure in relation to telomere function in living cells. The program contains three distinct projects: 1.1 Is there a role for loss of telomere function in human leukemia development? Age-related loss of telomeric DNA may result in chromosome end-to-end fusions, and subsequent chromosome degradation and gross genomic instability. Using frozen samples of more than 300 Acute Myeloid Leukemia (AML) patients with well-characterized chromosomal aberrations, we study the association of critical telomere loss and specific chromosomal abnormalities. We have developed specific analysis methods to investigate length of telomeric DNA at individual chromosome ends in leukemic cells using dedicated image analysis computer programs. 1.2 Which factors are critical in regulating telomerase-mediated telomere elongation? Telomerase, the major telomere elongating mechanism that is active in hematopoietic stem cells and most cancer cells, regulation is studied in stem cells and in a model of >20 telomerase-reconstituted fibroblast clones. The model allows investigation of mechanisms in telomerase mediated telomere elongation. We have identified the in vitro activity of telomerase as the major regulator of telomere length in these telomerase reconstituted fibroblasts. Unexpectedly, the activity of telomerase is not related to expression levels of its two known components. The results indicate the presence of major, as yet unknown modulators of the activity of the telomerase enzyme complex. Current research is focussed identification of these modulators, using the model of previously generated telomerase-reconstituted fibroblast clones. Preliminary results of large scale array screens (Affymetrix) have been obtained and are being analyzed. 1.3 Telomeric protein organization in living cells. Effective chromosome end-protection and telomerase activation requires the presence of specific telomeric DNA-binding proteins. The molecular mechanisms how telomeric proteins are involved in chromosome end-protection and telomerase recruitment are largely unknown. Elucidation of these molecular mechanisms may be critical in developing future strategies to modulate telomere function in cancer and aging. Telomere organization in living cells is studied using Fluorescent-Protein-tagging of telomeric proteins and confocal microscopy with bleaching and Fluorescence Resonance Energy Transfer (FRET). Sofar, our results have indicated distinct telomere association patterns of the three human telomeric DNA binding proteins that have currently been identified. The results suggest the presence of distinct nucleoprotein complexes in the two major telomere functions, i.e. telomerase activity regulation and protection of the ultimate chromosome end against end-to-end fusion.

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Betrokken personen

Projectleider Dr. J.M.J.M. Zijlmans

Classificatie

D21300 Biochemie
D21400 Genetica
D21500 Histologie, celbiologie

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