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Rel 2.9 - 29th January 1998



Extension of Life-Span by Introduction of Telomerase into Normal Human Cells

Science, Jan 16, (1998).

Andrea G. Bodnar, Michel Ouellette, Maria Frolkis Shawn E. Holt, Choy-Pik Chiu, Gregg B. Morin, Calvin B. Harley, Jerry W. Shay, Serge Lichtsteiner, Woodring E. Wright

Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and fore-skin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced staining for b-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.

Short telomeres on human chromosome 17p.

Nat Genet 1998 Jan;18(1):76-80

Martens UM, Zijlmans JM, Poon SS, Dragowska W, Yui J, Chavez EA, Ward RK, Lansdorp PM

Terry Fox Laboratory for Hematology/Oncology, British Columbia Cancer Research Centre, Vancouver, Canada.

Human chromosomes terminate in a series of T2AG3 repeats, which, together with associated proteins, are essential for chromosome stability. In somatic cells, these sequences are known to be gradually lost through successive cells divisions; however, information about changes on specific chromosomes is not available. Individual telomeres could mediate important biological effects as was shown in yeast, in which loss of a single telomere results in cell-cycle arrest and chromosome loss. We now demonstrate by quantitative fluorescence in situ hybridization (Q-FISH; ref. 7) that the number of T2AG3 repeats on specific chromosome arms is very similar in different tissues from the same donor and varies only to some extent between donors. In all sixteen individuals studied, telomeres on chromosome 17p were shorter than the median telomere length--a finding confirmed by analysis of terminal restriction fragments from sorted chromosomes. These observations provide evidence of chromosome-specific factors regulating the number of T2AG3 repeats in individual telomeres and raise the possibility that the relatively short telomeres on chromosome 17p contribute to the frequent loss of 17p alleles in human cancers.

Telomerase activity and telomere length in pediatric patients with malignancies undergoing chemotherapy.

Leukemia 1998 Jan;12(1):13-24

Engelhardt M, Ozkaynak MF, Drullinsky P, Sandoval C, Tugal O, Jayabose S, Moore MA

James Ewing Laboratory of Developmental Hematopoiesis, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

Telomerase activity and telomere length in mononuclear cells (MNCs) and granulocytes from peripheral blood (PB) and bone marrow (BM) specimens were studied in pediatric acute leukemia (ALL, n = 15; AML, n = 11) and pediatric solid tumor (ST) patients (n = 9) at diagnosis, during and after chemotherapy. In four ST patients, tumor tissue was also available. For comparative analysis, MNCs from healthy donors (n = 53) were analyzed. Telomerase was evaluated using a modified telomeric repeat amplification protocol (TRAP) assay, and telomere length by terminal restriction fragment (TRF) analysis. At diagnosis, high telomerase activity was detected in MNCs from all leukemia patients, which was similar to the activity from ST biopsy specimens. This exceeded by 10- to 20-fold the activity in PB MNCs from ST patients and healthy donors (P < 0.05). Granulocyte fractions lacked telomerase activity in all groups. BM MNCs in leukemia patients revealed a four-fold higher telomerase activity than PB (P = 0.005). After induction chemotherapy and response to treatment, telomerase activity decreased to borderline or undetectable levels in PB MNCs in leukemia (P < 0.01). Average telomeres in PB MNCs from pediatric patients were significantly longer (n = 25; 10.9 kbp) than telomeres in PB and BM MNCs from adult healthy donors (7.45 kbp) (P < 0.0001). At diagnosis, telomeres were shorter from BM compared to PB specimens in leukemia (P < 0.05), and two peak TRFs were observed corresponding to the malignant and normal cell clones. With the attainment of remission, the lower TRF peak, reflecting the leukemic population, was lost. In leukemia patients, mean TRFs increased on average 2.2 kbp after induction chemotherapy, but decreased thereafter on consolidation and maintenance chemotherapy (1 kbp). This was comparable to an average telomere loss of 1.2 kbp in PB specimens from ST patients after chemotherapy. In all patients, telomere loss in granulocytes as compared to MNCs was more pronounced with 1.8 vs 1 kbp, respectively (P = 0.014). Our results demonstrate that at diagnosis, telomerase was consistently and highly upregulated in BM and PB specimens in leukemia, decreased after induction therapy, and correlated with remission. BM specimens in leukemia had higher telomerase activity, probably due to the greater leukemic burden than in PB. Telomeres were significantly longer in children than in adults, but shortened as a consequence of chemotherapy with repeated cycles of hematopoietic regeneration. In acute leukemia, with the loss of the leukemic burden after induction chemotherapy, longer mean TRFs were found, a reflection of the repopulation with normal cells. Our findings suggest that telomerase activity may be useful in the management of childhood malignancies. The significance of telomere length shortening in pediatric patients undergoing chemotherapy and possible telomere regeneration after myelosuppressive treatment remain to be determined.

DNA topoisomerase II cleavage of telomeres in vitro and in vivo.

Biochim Biophys Acta 1998 Jan 7;1395(1):110-120

Yoon HJ, Choi IY, Kang MR, Kim SS, Muller MT, Spitzner JR, Chung IK

Department of Biology, Yonsei University, Seoul, South Korea.

In this work, we have analyzed the reactivity of DNA topoisomerase II with telomeric DNA both in vitro and in vivo. Topoisomerase II cleavage reactions were performed on the tandem repeats of telomeric DNA. Analysis of this DNA on sequencing gels revealed that DNA topoisomerase II is catalytically active in cleaving the telomere DNA repeat. The topoisomerase II cleavage site is 5'TTAGG*G3' (cleavage site marked by the asterisk) and since telomere DNA is a tandem array of the above sequence, topoisomerase cleavage sites could exist every six base pairs. Detection of topoisomerase II cleavages was strongly dependent upon one specific topoisomerase II poison, etoposide (VP-16). A number of other topoisomerase II poisons were tested but did not stimulate cleavage activity at the telomere repeat. We have also analyzed the association of endogenous topoisomerase II with chromosomal telomeric DNA in HeLa cells. The in vivo complex of enzyme (ICE) bioassay was used to isolate topoisomerase II-DNA covalent complexes. In consistence with in vitro cleavage data, endogenous topoisomerase II-telomeric DNA complexes were detected in only etoposide-treated HeLa cells.



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