Preferential maintenance of critically short telomeres in mammalian cells heterozygous for mTert

Abstract

Prolonged growth of murine embryonic stem (ES) cells lacking the telomerase reverse transcriptase, mTert, results in a loss of telomere DNA and an increased incidence of end-to-end fusions and aneuploidy. Furthermore, loss of only one copy of mTert also results in telomere shortening intermediate between wild-type (wt) and mTert-null ES cells [Liu, Y., Snow, B. E., Hande, M. P., Yeung, D., Erdmann, N. J., Wakeham, A., Itie, A., Siderovski, D. P., Lansdorp, P. M., Robinson, M. O. & Harrington, L. (2000) Curr. Biol. 10, 1459-1462]. Unexpectedly, although average telomere length in mTert(+/-) ES cells declined to a similar level as mTert-null ES cells, mTert(+/-) ES cell lines retained a minimal telomeric DNA signal at all chromosome ends. Consequently, no end-to-end fusions and genome instability were observed in the latest passages of mTert(+/-) ES cell lines. These data uncover a functional distinction between the dosage-dependent function of telomerase in average telomere-length maintenance and the selective maintenance of critically short telomeres in cells heterozygous for mTert. In normal and tumor cells, we suggest that telomerase activity insufficient to maintain a given average telomere length may, nonetheless, provide a protective advantage from end-to-end fusion and genome instability.

Figure 1

Flow-FISH analysis of telomeric DNA content in ES cell populations. Average relative telomere fluorescence upon continued passage (p1-p120) of wt, two independently derived mTert^+/− ES cell lines, and two mTert^−/− ES cell lines. Representative, average telomere fluorescence intensity at each passage is presented for each cell line and not averaged between samples because of variability in initial telomere lengths in each cell line.

Figure 2

FISH analysis of ES cell metaphase preparations. (Upper) Representative metaphase preparations of wild-type ES cells at p75 (+/+ p75), and mTert^+/− and mTert^−/− ES cells at p90 (+/− p90, −/− p90). (Lower) The same images were overexposed to demonstrate that mTert^+/− cells retain detectable telomere signals at all chromosome ends. Chromosome ends with no telomeric DNA signal (arrows) and end-to-end fusions (*) are indicated.

Figure 3

Q-FISH analysis of individual ES cell metaphase preparations. The respective genotype is indicated at the top, and the passage number is at the left. Data were accumulated by using approximately 10 metaphases for each histogram. The ‘events per category’, or number of telomeres within a given range of telomeric DNA intensities, was plotted against the telomere DNA signal intensity with arbitrary units (0 = no telomeric DNA signal, and increasing in increments of arbitrary telomere fluorescence units to 90). Average and median telomere lengths are shown for each histogram (ave, med, respectively). Note that the decrease in mean telomere length appears greater by Q-FISH than by Flow-FISH in the latest passages in mTert^−/− ES cells (compare p90 −/−, Fig. 1 and 3). We believe this difference could reflect higher nonspecific (i.e., nontelomeric) fluorescence with the Flow-FISH method, combined with reduced telomere DNA signal intensities in Q-FISH under the image conditions required to capture a wide variation in telomeric DNA signal in a near-linear range.

Figure 4

Telomerase activity levels in mTert^+/− ES cells. (Upper) Telomerase activity in wild-type or mTert^+/− ES cells. TRAP was performed for 20 PCR cycles on 1.0, 0.5, 0.25, and 0.125 μg of cell extracts prepared from wt and mTert^+/− ES cell extracts at the indicated passage numbers. An internal PCR standard for the TRAP is shown at bottom right with an arrow. The asterisk (*) indicates a nonspecific product in mouse extracts that is resistant to RNase A treatment. (Bottom) The telomerase extension products were normalized to internal standard with nih image quant analysis; SDs (error bars) were calculated based on three separate experiments.

Figure 5

Quantitation of mTert mRNA levels in wild-type. mTert^+/− and mTert-null ES cells at early passage (A), p50 (B), and late passage (C). The relative copies of mTert mRNA were calculated based on a standard curve obtained by using quantitative RT-PCR (Taqman) analysis and then normalized to the level of Gapdh mRNA in the corresponding samples. The passage number and genotype of each sample is indicated above. Average ratios of mTert to Gapdh are shown above each data bar. In the wt p50 sample, there were reproducibly decreased levels of Gapdh mRNA, leading to the apparently higher ratio of mTert/Gapdh (data not shown) (D). Analysis of telomerase activity levels in late passages of ES cells. The respective genotypes and passage numbers are shown above. TRAP was performed for 20 cycles on 1.0, 0.5, 0.25, and 0.125 μg of cell extracts of the indicated genotype. An internal PCR standard for the TRAP is shown at bottom left with an arrow. The asterisk (*) indicates a nonspecific product in mouse extracts that is resistant to RNase A treatment.