Five dysfunctional telomeres predict onset of senescence in human cells

Abstract

Replicative senescence is accompanied by a telomere-specific DNA damage response (DDR). We found that DDR+ telomeres occur spontaneously in early-passage normal human cells and increase in number with increasing cumulative cell divisions. DDR+ telomeres at replicative senescence retain TRF2 and RAP1 proteins, are not associated with end-to-end fusions and mostly result from strand-independent, postreplicative dysfunction. On the basis of the calculated number of DDR+ telomeres in G1-phase cells just before senescence and after bypassing senescence by inactivation of wild-type p53 function, we conclude that the accrual of five telomeres in G1 that are DDR+ but nonfusogenic is associated with p53-dependent senescence.

Figure 1

Spontaneous telomere dysfunction in normal human cells. (A) Phase-contrast microscopy and meta-TIF assay images of Fre 74s-1 cells. Meta-TIF assay samples were stained with DAPI (blue), γ-H2AX IF (red) and telomere FISH (green). White arrows: meta-TIFs; black bars: 50 μm; white bars: 10 μm. (B) Growth curves. (C) Meta-TIF assay results (mean±s.d., n=3). (D) Percentage of γ-H2AX TIF-positive nuclei after treatment with 20 ng ml⁻¹ colcemid for 4 h, mitotic shake-off, plating and fixation at indicated times (mean±s.d., n=3 experiments quantifying 100 nuclei). (E) Meta-TIF assay results after indicated colcemid treatments (average±range, n=2; P, Student t-test). DAPI, 4′,6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; γ-H2AX, phosphorylated H2AX; IF, immunofluorescence; meta-TIF, metaphase telomere dysfunction-induced focus; PD, population doubling.

Figure 2

Five DDR+ telomeres precede replicative senescence. (A) Chromosome- and chromatid-type meta-TIFs as a percentage of total meta-TIFs (mean±s.d., n=3). Examples of chromatid- and chromosome-type meta-TIFs stained with DAPI (blue), γ-H2AX IF (red) and telomere FISH (green). (B) CO-meta-TIF assay results (mean±s.d., n=3) and examples stained with DAPI (blue), γ-H2AX IF (red) and telomere C-strand PNA FISH (green). (C) Flow cytometry of PI-stained dividing and senescent normal fibroblasts. (D) Calculated number of predicted G1-TIFs. CO, chromosome orientation; DAPI, 4′,6-diamidino-2-phenylindole; DDR, DNA damage response; FISH, fluorescence in situ hybridization; γ-H2AX, phosphorylated H2AX; IF, immunofluorescence; meta-TIF, metaphase telomere dysfunction-induced focus; PD, population doubling; PI, propidium iodide; PNA, peptide nucleic acid.

Figure 3

TIFs aggregate in senescent nuclei. (A) Senescent nuclei stained with DAPI (blue), γ-H2AX IF (red) and Chk2-Thr68, NBS1 or MRE11 IF (green). (B) Senescent nuclei stained with DAPI (blue), γ-H2AX IF (red) and telomere FISH (green). Images shown are a single deconvolved focal plane. Enlargements are shown at the right. White bars in (A) and (B): 5 μm. (C) Relative telomeric fluorescence intensity in 11 Fre 102s-3 nuclei within a circle drawn around the boundaries of a γ-H2AX focus (red circle) and the same circle around each of the 10 brightest (grey triangles) and 10 faintest (blue squares) telomeres in the same nuclei. DAPI, 4′,6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; γ-H2AX, phosphorylated H2AX; IF, immunofluorescence; TIF, telomere dysfunction-induced focus.

Figure 4

Relationship between telomere length and meta-TIFs. (A) Percentage of meta-TIFs with detectable (+) or undetectable (−) telomere FISH signals (mean±s.d., n=3). (B) Comparison of relative fluorescence intensity of γ-H2AX IF (red) and telomere FISH (green). (C) Quantification of relative telomeric fluorescence intensity between the γ-H2AX-positive and -negative sister telomeres of a chromosome-type meta-TIF (mean±s.d., n=3). (D) Cyto-centrifuged chromosomes stained with DAPI (blue), γ-H2AX IF (red) and TRF2 or RAP1 IF (green). DAPI, 4′,6-diamidino-2-phenylindole; DDR, DNA damage response; FISH, fluorescence in situ hybridization; γ-H2AX, phosphorylated H2AX; IF, immunofluorescence; meta-TIF, metaphase telomere dysfunction-induced focus; PD, population doubling; Tel, telomere.

Figure 5

Telomere dysfunction during lifespan extension and crisis. (A) Meta-TIF assay images from simian virus 40 LT-transfected cells (Fre 80/4Tii) at PD 28 and 45. (B) Growth curves. (C) Meta-TIF assays (average±range, n=2; except for Fre 80/4Tii PD 45, n=1). (D) Contribution of chromosome- and chromatid-type meta-TIFs (average±range, n=2). (E) Calculated number of G1-TIFs. (F) Calculated number of G1-TIFs from meta-TIF data for 17 immortalized cell lines (Cesare et al, 2009). (G) Representative images of standard cytogenetic preparations from Fre 80 and Fre 80/4Tii stained with telomere (red) and centromere (green) FISH; examples of fused chromosomes are indicated by white arrows. DAPI, 4′,6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; LT, large T-antigen; meta-TIF, metaphase telomere dysfunction-induced focus; PD, population doubling.