Elevated telomere-telomere recombination in WRN-deficient, telomere dysfunctional cells promotes escape from senescence and engagement of the ALT pathway

Abstract

Werner Syndrome (WS) is characterized by premature aging, genomic instability, and cancer. The combined impact of WRN helicase deficiency and limiting telomere reserves is central to disease pathogenesis. Here, we report that cells doubly deficient for telomerase and WRN helicase show chromosomal aberrations and elevated recombination rates between telomeres of sister chromatids. Somatic reconstitution of WRN function, but not a WRN helicase-deficient mutant, abolished telomere sister chromatid exchange (T-SCE), indicating that WRN normally represses T-SCEs. Elevated T-SCE was associated with greater immortalization potential and resultant tumors maintained telomeres via the alternative lengthening of telomere (ALT) pathway. We propose that the increased incidence of chromosomal instability and cancer in WS relates in part to aberrant recombinations between sister chromatids at telomeres, which facilitates the activation of ALT and engenders cancer-relevant chromosomal aberrations and tumor formation.

Figure 1.

G5 mTerc^-/- Wrn^-/- MEFs spontaneously immortalize and exhibit chromosomal aberrations. (A) Serial NIH 3T3 assays of six independently derived G5 mTerc^-/- Wrn^-/- cell lines passaged over 100 PDs. (B) Western blot analysis of p53, p21, p19, p16, and γH2AX expression in passage 1 (P1) to P5 parental (par), and immortalized clones 1 and 2 with (+) or without (-) 5 Gy γ-radiation (IR) treatment. Actin was used as loading control. (C) Chromosomal aberrations in P4 parental and immortalized clones. (Arrows) p-p arm chromosomal fusions; (arrowhead) chromatid break; (dc) dicentric chromosome. (D) Spectral karyotyping (SKY) of representative immortalized clone. (Arrow) p-p arm chromosomal fusions; (NRT) nonreciprocal translocations. (E) Quantitation of the number of chromosomal fusions per metaphase in the indicated cell lines as a function of increased passages in vitro.

Figure 2.

Characterization of telomere structure and TDMs in G5 mTerc^-/- Wrn^-/- immortalized clones. (A) Anaphase bridge formation (arrows) in immortalized clones. (B) Quantification of number of anaphase bridges per metaphase in cells of indicated genotypes. Error bars represent standard error of the mean (SEM). Asterisk (^⋆) indicates a p value of <0.001 (Student's t-test). (C) Metaphase chromosomes isolated from representative immortalized clones were stained by DAPI (blue) and telomeric DNA was detected by FISH with rhodamine-conjugated (CCCTAA)₃ PNA probe (red). Sites of chromosome fusions are indicated by arrowheads, and TDMs are labeled by arrows. (Panels a-c) TDMs from three independently immortalized clones. (Panel d) TDM from V-sarcoma. (D) Analysis of G-rich single-strand overhangs and telomere lengths from parental and immortalized clones at the indicated passages. A CHEF gel resolved digested DNAs, and in-gel hybridization with end-labeled (CCCTAA)₄ oligonucleotides was used to detect the 3′ G-strand overhangs. The same gel was then denatured and hybridized with (TTAGGG)₄ oligonucleotides to detect duplex telomeric DNA. Molecular weights of marker DNA are indicated.

Figure 3.

T-SCE is elevated in G5 mTerc^-/- Wrn^-/- cells. (A) Schematic of CO-FISH analysis (Bailey et al. 2004). (B) CO-FISH on G5 mTerc^-/- Wrn^+/- MEFs reveals two characteristic CO-FISH telomere signals per chromosome. (C) CO-FISH of G5 mTerc^-/- Wrn^-/- MEFs. T-SCEs are abundant, as exhibited by the split (double) telomere-signal hybridization patterns. Note Robertsonian translocation without telomere sequence at the point of fusion (arrowhead). (D) Quantification of the average number of T-SCEs per chromosome detected in cells of the indicated genotypes. Error bars represent standard error of the mean (SEM). Asterisk (^⋆) indicates a p value of <0.05 (Student's t-test). (E) Genomic SCE in G5 mTerc^-/- Wrn^-/- MEFs. Harlequin anti-BrdU staining pattern after two rounds of incorporation reveals genomic SCE levels are not elevated. (F) Dual labeling experiment: CO-FISH with telomere (T-SCEs, red signals) and mouse major satellite (single-sided, green signal) probes in G5 mTerc^-/- Wrn^-/- MEFs; SCE within this highly repetitive block of pericentromeric repeats is not elevated. (G) Reconstitution of mutant p53^R175H activity in two independent G3 mTerc^-/- Wrn^-/- MEF clones. p53 and p21 levels were not elevated following 10 Gy IR treatment. (H) T-SCE levels in p53^R175H-immortalized G3 mTerc^-/- Wrn^-/- MEF clones. Error bars represent standard error of the mean (SEM). p values indicate a lack of significance (Student's t-test).

Figure 4.

WRN helicase activity is required to suppress T-SCEs. (A) Western blot of G5 mTerc^-/- Wrn^-/- cell lines reconstituted with vector, human WRN, hWRN-K557M, and hWRN-E84A. Actin was used as loading control. (B) Wild-type mouse and human WRN, but not vector, rescues the growth defect observed in G5 mTerc^-/- Wrn^-/- MEFs. Error bars represent standard error of the mean (SEM). (C) Quantitation of STL in two immortalized cell lines reconstituted with vector, hWRN, hWRN-K557M, or hWRN-E84A. Error bars represent standard error of the mean (SEM). Asterisk (^⋆) indicates a p value of <0.001 (Student's t-test). (D) Quantitation of T-SCE levels following reconstitution with the indicated proteins in either immortalized or parental G5 mTerc^-/- Wrn^-/- MEFs. Error bars represent standard error of the mean (SEM). (^⋆) p < 0.001 (Student's t-test). (E) Western blot of G5 mTerc^-/- Wrn^-/- parental or immortalized cell lines with vector or mouse WRN. Actin was used as loading control. (F) T-SCEs are reduced in mWRN, but not vector-reconstituted cell lines. (Arrows) Chromosomes containing T-SCEs. (G) Quantitation of reduction of T-SCE levels in G5 mTERC^-/- Wrn^-/- parental cell lines following reconstitution with mWRN. Error bars represent standard error of the mean (SEM). (^⋆) p < 0.001 (Student's t-test).

Figure 5.

Transformation and tumorigenic potential of immortalized G5 mTerc^-/- Wrn^-/- MEFs. (A) Tumorigenicity of injected cell lines. pBabe-H-Ras^V12 (top) and pBabe-vector transfected (bottom) immortalized MEFs were injected into flanks of SCID mice (1 × 10⁶ cells per injection site). Arrows point to resultant tumors. (B) H&E analysis of the tumors indicates that they are poorly differentiated sarcomas. (C) Chromosomal aberrations in SCID tumors. Metaphase spreads from H-Ras^V12 (top) and V-sarcoma (bottom), which were stained by DAPI (blue) and rhodamine-conjugated (CCCTAA)₃ PNA probe (red). Fused chromosomes are indicated by arrows. (Y-axis) Frequency; (X-axis) telomere fluorescence units (TFUs). (D) Telomere length analysis by Q-FISH in H-Ras^V12 sarcoma (top), V-sarcoma (middle), and immortalized parental (bottom) cell lines.

Figure 6.

Engagement of the ALT pathway in H-Ras^V12 and V-sarcomas. (A) TRF Southern analysis for telomere lengths of indicated samples. End-labeled (TTAGGG)₄ oligonucleotides were used to detect the 3′ G-strand overhangs, and duplex telomeric DNA was detected with end-labeled (CCCTAA)₄ oligonucleotides. Note the increase in telomere length heterogeneity in both SCID tumor cell lines. (B) Colocalization of PML and TRF1 at telomeres. PML and TRF1 colocalized only in a subset of cells containing ALT foci (arrows). (C) Quantitation of ALT-associated PML bodies in the indicated cell types. Error bars represent standard error of the mean (SEM). (^⋆) p < 0.01.