Abstract
Telomere length can be maintained by telomerase or by a recombination-based pathway. Because individual telomeres in cells using the recombination-based pathway of telomere maintenance appear to periodically become extremely short, cells using this pathway to maintain telomeres may be faced with a continuous state of crisis. We expressed telomerase in a human cell line that uses the recombination-based pathway of telomere maintenance to test whether telomerase would prevent telomeres from becoming critically short and examine the effects that this might have on the recombination-based pathway of telomere maintenance. In these cells, telomerase maintains the length of the shortest telomeres. In some cases, the long heterogeneous telomeres are completely lost, and the cells now permanently contain short telomeres after only 40 population doublings. This corresponds to a telomere reduction rate of 500 base pairs/population doubling, a rate that is much faster than expected for normal telomere shortening but is consistent with the rapid telomere deletion events observed in cells using the recombination-based pathway of telomere maintenance (Murnane, J. P., Sabatier, L., Marder, B. A., and Morgan, W. F. (1994) EMBO J. 13, 4953-4962). We also observed reductions in the fraction of cells containing alternative lengthening of telomere-associated promyelocytic leukemia bodies and extrachromosomal telomere repeats; however, no alterations in the rate of sister chromatid exchange were observed. Our results demonstrate that human cells using the recombination-based pathway of telomere maintenance retain factors required for telomerase to maintain telomeres and that once the telomerase-based pathway of telomere length regulation is engaged, recombination-based elongation of telomeres can be functionally inhibited.
Highlights
Telomeres shorten due to the inability of lagging C-strand synthesis to replicate the extreme ends of linear DNA [2] as well as nuclease processing of the parental C-strand following leading strand synthesis [3, 4]
The average telomere length declined after 40 population doublings from 25 kb to 6.2 kb for clone 1 and 2.6 kb for clone 2 (Fig. 2B). This rate of shortening of about 500 bases/population doubling is 10-fold faster than the rate of telomere loss observed for normal human cells. This rapid loss is consistent with the observed rapid deletion of long telomeres that occurs in yeast and human cells using alternative lengthening of telomeres (ALT) [1, 28]
The distribution pattern of the telomere lengths observed in VA13hTel clones 1 and 2 is similar to the distributions found in most cancer or in vitro immortalized cells using telomerase to maintain telomeres
Summary
TRF and TRAP Analysis—Telomere length was determined using TRF analysis. First, cell pellets were suspended in 100 mM NaCl, 100 mM EDTA (pH 8.0), and 10 mM Tris (pH 8.0), using 30 l/106 cells. The slides were incubated with a hybridization mixture (20 l) containing 70% formamide, 3Ј-Cy3-conjugated (CCCTAA)3 2Ј-deoxyoligonucleotide N3Ј-P5Ј phosphoramidate probe [27], 0.25% (w/v) blocking reagent (1096 176; Roche Molecular Biochemicals), and 5% MgCl2 in 10 mM Tris (pH 7.2) for 3 min at 78 °C. The slides were incubated for 3 h at room temperature and washed with 70% formamide, 0.1% bovine serum albumin, and 10 mM Tris (pH 7.2) four times (15 min each time). The slides were washed with 0.15 M NaCl, 0.05% Tween-20, and 0.05 M Tris four times (5 min each time); dehydrated by a 2-min incubation in 70% ethanol, a 2-min incubation in 90% ethanol, and a 2-min incubation in 100% ethanol; air dried in the dark; mounted with Vectashield containing DAPI (Vector Laboratories); and imaged using a Zeiss Axioplan 2 microscope. The cells were blocked with a solution of 3% bovine serum albumin/PBS
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
