Dynamics of telomere erosion and its association with genome instability in myelodysplastic syndromes (MDS) and acute myelogenous leukemia arising from MDS: a marker of disease prognosis?
Introduction
During the last decade, both the telomeres—ends of human chromosomes and enzyme telomerase have been subjected to intensive investigation as potential molecular markers important for diagnosis and prognosis of almost all neoplasms including leukemia (reviewed in [1], [2], [3], [4], [5], [6], [7], [8] recently published).
Telomeres represent structural and functional units composed of protein and non-coding repetitive DNA sequences (TTAGGG)n. The role of telomeres is to ensure the integrity of chromosomes and to protect them from degradation, recombination and from a loss of DNA sequences. Physiological long-term telomere shortening in peripheral blood cells by roughly 40–80 bp per year [9] is supposed to be enhanced during malignant transformation. Rapid erosion of telomeres results in an extensive DNA instability. Once a length of 5–7 kbp has been reached, genomic instability gradually progresses until a critical length of 3–4 kbp, which results in rapid elimination of altered cells. However, a small number of cells may escape from this “mortality phase” and give rise to a clone with unlimited proliferative potential [9]. This is almost always accomplished by activation of ribonucleoprotein telomerase to maintain the function of telomeres by synthesizing telomeric repeats. Both reactivation of telomerase as well as its increased expression leads to elongation or stabilization of telomeres and hence to immortalization of cells with genetic damage [10]. An intensive study of pathways responsible for regulation of telomere length and telomerase activity might elucidate a molecular basis of malignant transformation of cells and may serve for therapeutic approaches targeting telomere–telomerase complex and affecting “immortality” of telomerase expressing malignant cells (reviewed in [11], [12]).
Hematopoietic cells of patients with the myelodysplastic syndromes (MDS) constitute a valid object for investigation of molecular mechanisms preceding malignant transformation and development of overt leukemia. MDS represents a heterogeneous group of clonal disorders of multipotent hematopoietic stem cells characterized by ineffective hematopoiesis connected with peripheral blood cytopenia, bone marrow morphological abnormalities and with tendency to progress into acute leukemia in approximately one third of cases.
Despite a considerable progress bringing new insights into the etiology and pathogenesis of MDS, the molecular background of MDS evolution towards leukemia has not been entirely elucidated yet. A modified dynamics of telomere–telomerase complex may be one of the critical factors playing an important role in this process. A rapid proliferation of hematopoietic stem cells in MDS patients may lead to an accelerated telomere shortening and consequently to genome instability.
We have focused on evaluation of telomere length in bone marrow and peripheral blood cells obtained from patients with MDS and acute myelogenous leukemia (AML). The aim of our study was to find out, whether a rapid telomere shortening is associated with the progression of MDS from early phases to advanced phases and subsequently towards acute leukemia. We investigated a possible relationship between telomere length and leukemic progression by comparing the results obtained in different MDS risk groups evaluated according to International Prognostic Scoring System (IPSS). We also studied relations between telomere erosion and genome instability manifested by chromosomal aberrations, mainly by complex rearrangements of karyotype.
Section snippets
Patients and samples
Molecular analyses were performed on mononuclear bone marrow and peripheral blood cells of 52 samples of 50 patients with FAB subtypes of primary myelodysplastic syndromes and acute myelogenous leukemia arising from MDS. Moreover, 21 patients with untreated primary AML (FAB subtypes M0–M4) were also investigated. Majority (66) of investigated patients were adults at the age ranging from 21 to 80 years () treated at the Institute of Hematology and Blood Transfusion in Prague.
Methods
Mononuclear cells obtained from bone marrow and peripheral blood samples were separated by Ficoll-Paque density gradient centrifugation. High molecular weight genomic DNA was extracted by modification of the salting-out method according to the standard protocol.
Telomere lengths were measured by terminal repeat fragment (TRF) method performing non-radioactive chemiluminescent assay. The method is based on Southern blot hybridization with the oligonucleotide labeled by digoxigenine containing
Dynamics of telomere length in MDS and AML
On the base of our results of TRF of healthy age-matched donors (, range 7.16–9.82), we postulated cases with TRF shorter than 7.5 kbp as cases with reduced telomeres, similarly as in work of Boultwood et al. [16].
The results on TRFs, karyotyping and stratifying of MDS patients according to the IPSS are showed in Table 1, Table 2. A considerable heterogeneity of telomere length, defined as terminal restriction fragment (TRF) was found in bone marrow cells of MDS patients
Discussion
Only small number of studies trying to elucidate dynamics of telomere shortening in preleukemic and leukemic cells and its association with clinical status and with frequency of chromosomal aberrations have been published (reviewed in [18]). Our findings confirmed an anticipated tendency of accelerated telomere erosion connected with progression of MDS towards overt leukemia, described by some authors [16], [19]. Abnormal telomere reduction was already present in approximately one third of our
Acknowledgements
This study was supported by the grants of IGA MZ CR: NC/5903-3, NC/7606-3, NK/7713-3, 237360001, and GACR 301/01/0200.
Contributions. (1) Conception and design: Z. Sieglová. (2) Analysis and interpretation of data: S. Žilovcová, Z. Sieglová, J. Čermák, K. Michalová, H. Řihová, R. Dvořáková, J. Březinová, Z. Zemanová. (3) Drafting the article: Z. Sieglová. (4) Critical revision of the article for important intellectual content: J. Čermák, K. Michalová. (5) Final approval of article: Z. Sieglová.
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