Aneuploidy is a fundamental principle of many cancer cells and is mostly related to defects in mitotic segregation of chromosomes. Many solid tumors as well as some preneoplastic lesions have been shown to contain polyploid chromosome numbers. The exact mechanisms behind whole-genome duplications are not known but have been linked to compromised mitotic checkpoint genes. We now report that the telomere checkpoint plays a key role for polyploidy in colon cancer cells. Telomerase suppression by a dominant-negative mutant of hTERT and consecutive telomere dysfunction in wild-type HCT116 colon cancer cells resulted in only minor stable chromosomal alterations. However, higher ploidy levels with up to 350 chromosomes were found when the cell-cycle checkpoint proteins p53 or p21 were absent. These findings indicate that telomere dysfunction in the absence of cell-cycle control may explain the high frequency of alterations in chromosome numbers found in many solid tumors.
Numerical chromosomal aberrations, referred to as aneuploidy, are commonly observed in human cancer (Sen, 2000; Rajagopalan and Lengauer, 2004). Although it is not clear whether chromosomal instability occurs in the early or late stages of tumor development, there is agreement that aneuploidy must be viewed as a driving force in oncogenesis (Hede, 2005). The mechanism and molecular determinants of chromosomal instability are not yet clearly understood (Bharadwaj and Yu, 2004; Rajagopalan and Lengauer, 2004). However, recent research has indicated that defects in the spindle checkpoint, a surveillance mechanism crucial for the proper segregation of chromosomes during cell division, may promote aneuploidy and tumorigenesis. The disruption of several cell-cycle genes has been shown to contribute to defective segregation of chromosomes as is the case with mutations of the mitotic checkpoint gene BUBR1 (Hanks et al., 2004), the breast cancer predisposing genes BRCA1 (Wang et al., 2004) or BRCA2 (Daniels et al., 2004) and the p53-dependent tumor suppressor gene hCDC4 (Rajagopalan et al., 2004). Interestingly, mitotic defects in aneuploid tumor cells were first described by Hansemann (1891), more than a century ago, who even discovered two additional chromosomal structures such as the formation of anaphase bridges and multipolar mitoses. As anaphase bridging is known to be a result of telomere erosion and chromosome end-to-end fusion (Maser and DePinho, 2002), the exact role of telomere function in the context of aneuploidy remains unclear. Here, we report that two common features of cancer cells, loss of telomere function and disruption of cell-cycle control, are capable of triggering extensive chromosomal instability in colon cancer cells.
To investigate the role of the telomere checkpoints (Feldser et al., 2003) in the context of telomere dysfunction and genomic instability, we analysed the phenotypic effects of telomerase inhibition in the well-defined colon cancer cell line HCT116 and its variants carrying inactivated p53 or p21. The HCT116 cell line has been described to contain a near-diploid chromosome content and has intact damage- and spindle-dependent checkpoints (Bunz et al., 1998). Telomere dysfunction was induced by ablation of telomerase activity using retroviral transduction with a dominant-negative (DN) mutant of hTERT (pOS-DN-hTERT-IRES-GFP). After 4–5 weeks of cell culture, positively transduced cells were individually propagated and three control clones transfected with the pOS-IRES-GFP vector (pOS) as well as a total of 10 single cell clones (DN) in p53+/+ (n=3), p21−/− (n=3) and p53−/− (n=4) were selected for further analysis. In contrast to the moderate telomerase activity, which was similar among the three control (pOS) clones and also found in the non-infected parental cells, all DN-transduced cells lacked telomerase activity as measured by TRAP assay (Figure 1).
As expected, all telomerase-negative clones (n=10) showed no immediate effect on growth potential, but stopped proliferation after a lag period of 40–65 cumulative population doublings (CPDLs) (Figure 2a–c). Accordingly, the presence of p53 or p21 did not influence the antiproliferative effect in general. However, the three telomerase-negative clones with wild-type p53 stopped proliferation uniformly after 40 CPDLs (Figure 2a), whereas the three p21−/− and, particularly, the four clones with lack of p53 showed a larger variability in their remaining population doublings (Figure 2b and c). Growth arrest was accompanied in all subclones by typical morphological changes of senescent cells such as enlarged and flattened size and vacuolated cytoplasm (Figure 1d). Unexpectedly, none of all telomerase-negative clones showed senescence-associated β-galactosidase activity (Figure 2d). Instead, a gradual increase in apoptosis with up to 43% was found in late passaged cells (see Materials and methods). This was indicated by accumulation of sub-G1 cells in the cell-cycle analysis (Figure 3a). Furthermore, p53+/+ cells showed an increased number of G0–G1 cells near the end of their lifespan, whereas p53−/− and p21−/− cells accumulated in G2–M phase (Figure 3b).
The mean telomere length was similar between the parental cell lines measuring approximately 6.1±0.5 kb for p53+/+ cells, 6.3±0.4 kb for p53−/− and 7.1±0.2 kb for p21−/− cells as analysed by flow fluorescence in situ hybridization (flow-FISH) (n=5). Upon the end of cell culture, the mean telomere length in DN cells was found to be between 5.0±0.2 and 5.6±0.4 kb (data not shown). Furthermore, telomere erosion was analysed by quantitative fluorescence in situ hybridization (Q-FISH) (Table 1), which revealed that the mean telomere length was significantly shorter (P<0.005, indicated by ** in Table 1) in all clones with absent telomerase activity at later passages as compared to control cells expressing telomerase.
Concomitant with the increased rate of telomere erosion, we found signs of telomere dysfunction such as an increase of end-to-end fusions (EEF) and signal-free ends (SFE) in the DN cells. Interestingly, the level of telomere dysfunction was significantly elevated in p21−/− and especially p53−/− cells. Signal-free ends increased from 7.50±3.62 per metaphase in p53+/+ up to 14.94±7.98 in p53−/− clones per metaphase (twofold, P<0.005; Table 1), and EEF from 0.26±0.44 per metaphase (in p53+/+ clones) to maximum 3.50±3.29 (in p53−/− clones) per metaphase (>10-fold, P<0.005; Table 1). Furthermore, the different levels of telomere dysfunction were accompanied by striking alterations in chromosome numbers. Whereas all telomerase-deficient clones had a subset of metaphases with reduced chromosome number compared to the control clones, an increase in hyperdiploidy as well as polyploidy was additionally found in cells with defective p53 and p21 (e.g. chromosome range in clone p53−/− DN1: 30–350).
To determine a potential mechanism for these numerical aberrations, we investigated the degree of centrosome amplification by using immunofluorescence with an antibody against γ-tubulin (Fukasawa, 2002). A slightly elevated level of cells with four instead of two centrosomes was already found in pOS-transduced p53−/− and p21−/− cells (8 and 11%, respectively) compared to p53+/+ cells (3%) (Figure 4a). However, this centrosome number aberrations were highly increased upon telomere dysfunction reaching 19% in p21−/− and 24% in p53−/− cells. Furthermore, we even found interphase nuclei (Figure 4b) and metaphases (Figure 4c) with up to six centrosomes, indicating a high level of defective mitotic segregation and multipolar mitosis (Figure 4e and f). In addition, the formation of anaphase bridges was determined (Figure 4d–f). The anaphase bridge index (ABI) was increased in all clones with telomere dysfunction, reaching even a slightly higher level in p53−/− and p21−/− cells than in p53 wild type (wt) cells (P=0.049).
As anaphase bridges are a sign of genomic instability causing breakage of the chromosome during mitosis and consequently rearrangements during the following cell cycles (reviewed in Murnane and Sabatier, 2004), multiplex (M)-FISH analyses were performed in HCT116 cells with functional and dysfunctional p53 status (Figure 5 and Table 2). These studies clearly demonstrated that telomerase activity in the presence or absence of p53 did not significantly influence the genetic make up of the cells. Both cell types only showed few different stable karyotypic characteristics that were all derived from a common precursor population (t(8;16), t(10;16) and t(17;18)) in the p53+/+ cells, whereas the p53−/− cells contained two additional stable aberrations (del(5q) and t(5;7)). Furthermore, they both showed only nine sporadic aberrations (0–2 extra aberrations per metaphase) in the 10 M-FISH karyotypes analysed each. On the other hand, lack of telomerase did affect both cell types significantly. This did not result in a high frequency of stable translocations (Figure 5a and b) but manifested in a high frequency of sporadic aberrations (0–6 per metaphase) in the p53+/+ DN-hTERT cells with a total of 27 per 10 metaphases and a total of 33 per 10 metaphases (1–7 per metaphase) in the p53−/− DN-hTERT cells. These aberrations also included a high number of fusions (14 and 6, respectively) not seen in any of the telomerase-positive variants (Figure 5c and d). The telomerase-positive p53+/+ and p53−/− cells also demonstrated numerical stability (Table 2). All 14 telomerase-positive p53+/+ cells were diploid. Also only one out of 18 metaphases derived from the p53+/+ DN-hTERT cells was hyperdiploid (n=64). Importantly, the same low frequency was seen for the telomerase-positive p53−/− cells. Only one out of 14 metaphases was hyperdiploid (n=60). However, 20% (five out of 21) of the 53−/− DN-hTERT cells showed high chromosome numbers (n=84, 82, 96, 81, 121). Thus, short telomeres in combination with loss of the p53 checkpoint clearly favored numerical changes.
Two major mechanisms that are essential to prevent chromosomal instability have been described (Rajagopalan and Lengauer, 2004). One regulates the separation of sister chromosomes through activation of the mitotic checkpoint, which is governed by genes such as BUBR1 and Mad2, and the other modulates the attachment of chromosomes to spindle microtubules requiring the activity of Aurora kinases. The data presented here extend this knowledge suggesting that telomere dysfunction appears to play a key role in defective mitotic segregation of chromosomes. Importantly, this effect is primarily dependent on the loss of p53 or p21 checkpoint function.
Although there is growing body of evidence, which suggests that defects in the spindle checkpoint are major factors for chromosomal instability (Jallepalli and Lengauer, 2001), mutation of mitotic checkpoint genes has not been found very frequently (Cahill et al., 1998; Shichiri et al., 2002; Hanks et al., 2004). Interestingly, the molecular mechanisms presented here, namely loss of p53 function (Vogelstein et al., 2000) and short dysfunctional telomeres (de Lange, 1995), represent one of the most common abnormalities in human cancer cells, which therefore could explain their frequent chromosomal instability. Although the expression of telomerase might restore telomere function in the majority of tumor cells during evolution from normal to malignant cells, critical telomere shortening may initially induce loss of telomere function and chromosomal instability in precursor cells as indicated here from the results with the telomerase-deficient p53+/+ and p53−/− cells as well as recently demonstrated in patients with colitis ulcerosa (O'Sullivan et al., 2002). Similarly, a peak in ABI has been reported to occur in early high-grade dysplastic lesions and less in more advanced carcinoma stages (Rudolph et al., 2001). Furthermore, our finding is in agreement with a report from Filatov et al. (1998), who described that chromosomal stability depends on telomere maintenance in in vitro aging fibroblasts, which express the E6 oncoprotein.
It has been speculated that anaphase bridging can be resolved by asymmetric segregation, which results either in loss of chromosomes induced by a break in the spindle or terminal deletion caused by a break in the chromatid (de Lange, 1995). In addition to this, our data suggest an even more complex picture that depends on p53 or p21 checkpoint status (Figure 6): whereas functional p53 pathway appears to initiate a DNA damage cascade following anaphase bridging and telomere dysfunction (Herbig et al., 2004), absence of this cell-cycle checkpoint leads to endoreduplication (Bunz et al., 1998; Edgar and Orr-Weaver, 2001) and most likely to cytokinesis failure, which ultimately results in centrosome amplification and polyploidy (Gisselsson and Hoglund, 2005). Such supernumerary centrosomes can lead to spindle multipolarity (see Figure 4f), which has been reported in many cancer cell types (Saunders et al., 2000). In support of this model, the M-FISH analysis did not reveal a high frequency of stable chromosomal rearrangements as expected from an ongoing chromosomal instability (‘bridge–breakage–fusion cycles’) but clearly demonstrated a higher frequency of additional aberrations and increased chromosome numbers. Interestingly, a very recent study demonstrated that anaphase bridging typically resulted in either centromeric chromatin fragmentation or centromere detachment, leading to pericentromeic chromosome rearrangements and loss of whole chromosomes (Stewenius et al., 2005).
In a mouse model, telomere dysfunction and mutation of p53 (mTerc−/− p53+/−) was shown to promote the development of epithelial cancers, which – different from our findings with loss of two p53 alleles – were associated with complex non-reciprocal translocations (Artandi et al., 2000). In general, it has to be taken into account that there are fundamental differences between mouse and human telomere regulation (Wright and Shay, 2000; Smogorzewska and de Lange, 2002). Therefore, this difference in structural chromosomal aberrations may be partly species specific. However, in the context of two defective p53 alleles, telomere dysfunction was also associated with a higher level of aneuploidy and accelerated carcinogenesis in mice (Chin et al., 1999), which confirms our data on human cells. Noteworthy, although the lack of tumor suppressor gene p53 and its downstream target p21 accelerates aneuploidy in our experimental system, targeted inactivation of p53 without telomere dysfunction does not result in aneuploidy in the HCT116 cell line as previously reported by Bunz et al. (2002).
In summary, this study underscores the important role of telomere dysfunction and concomitant cell-cycle checkpoint disruption in the generation of abnormal chromosome numbers. It will be interesting to examine if the telomere damage response pathway is directly involved in the regulation of mitotic checkpoint genes. Finally, alterations in chromosome numbers not only account for malignant transformation but also appear to contribute to aging phenotypes (Fernandez-Capetillo and Nussenzweig, 2004), thus making aneuploidy an essential mechanism of two important cellular processes.
Materials and methods
The HCT116 colon cancer cell lines were kindly provided by Dr B Vogelstein. The wt HCT116 form (HCT116 p53+/+ p21+/+) with normal expression of p53 and p21 was used, as well as a derivative in which both p21WAF1 alleles were disrupted by targeted homologous recombination (HCT116 p53+/+ p21−/−) and a derivative in which both p53 alleles were deleted in the same way (HCT116 p53−/− p21+/+) (Bunz et al., 1998). Cells were cultured in McCoy's 5A medium (Invitrogen, Karlsruhe, Germany) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen, Karlsruhe, Germany) and 1% of penicillin/streptomycin in a humidified 5% CO2 atmosphere.
Retrovirus production and infection protocol
Retroviral gene transfer of a DN mutant of hTERT was performed as recently described (Zimmermann et al., 2004; Pantic et al., 2005). HCT116 cells were infected with a DN-hTERT-IRES-GFP and a pOS GFP control vector. Single GFP-positive cells were cloned on a MoFlo high-speed cell sorter (Cytomation, Freiburg, Germany).
After the single cell proliferated to 80–90% of confluence in 96-well plates, cells were transferred into 24- or 48-well plates. The day of plating cells was designated as day 0. Early passaged are cells between day 0 and the beginning of lag phase on the cell proliferation curves (with different CPDL range: from 0 to 38 or 50). Late passaged cells are cells from the beginning of lag phase to the end of cell proliferation CPDL range from 38 to 57).
Cells were grown on six-well plates for 2 days, trypsinized, washed in phosphate-buffered saline (PBS) and fixed in 70% ethanol on ice. Then, the cells were stained with propidium iodide and cell-cycle distribution was measured on FACS can flow cytometer (Becton Dickinson, Heidelberg, Germany). Results were analysed using CellQuest (Becton Dickinson, Heidelberg, Germany) and Mod Fit software (Verity Software House Topsham, ME, USA). For each sample, 25 000 events were collected and aggregated cells were gated out. For the evaluation of apoptosis (sub-G1), all cells were analysed.
HCT116 cells were plated on chamber slides (Nalge Nunc Inter, Naperville, IL, USA) and senescence detection assay was carried out according to the manufacturer's protocol (Trevigen Inc., Gaitersburg, MD, USA).
To visualize centrosomes, staining procedures were performed as described (Borel et al., 2002) with some modifications. Briefly, cytospins were fixed with 2% paraformaldehyde in PBS at 37°C for 10 min and then permeabilized for 3 min with 0.2% Triton X-100 in PBS. Cells were incubated with primary anti-γ-tubulin antibodies (Clone GTU-88, Sigma-Aldrich, Steinheim, Germany) diluted 1:100 in PBS containing 0.1% Tween 20 and 3% BSA for 1 h at 37°C in a humid chamber. After three washes with PBS containing 0.1% Tween 20, cells were incubated for 30 min with TRITC-conjugated rabbit anti-mouse IgG secondary antibody (DAKO Cytomation, Denmark) (1:60). DNA was counterstained by the addition of 4,6-diamidino-2-phenylindole (DAPI) (Vysis Inc., Downers Grove, IL, USA) in mounting medium (Vectashield, Vector Laboratories Inc., Burlingame, CA, USA). Quantification was performed by manual counting on three representative fields (on 200–300 cells). Images were captured with confocal Leica, TCS SP2 AOBS microscope (Leica Microsystem, Wetzlar) supported by Axiovision 3.1 software.
For analysis of mitotic figures, cells on chamber slides were washed in PBS for 5 min, fixed in methanol:acetic acid (3:1) at −20°C for 30 min, air dried and stained with hematoxylin–eosin (H&E) or DAPI (Gisselsson et al., 2000). At least 30 anaphases and 50 metaphases were analysed on three different slide fields. Anaphase bridge index was determined by dividing the number of anaphases with bridges by the total number of anaphases (Gordon et al., 2003). Anaphase bridge was defined as one or more lines spanning two separating anaphase poles.
Cytogenetic and Q-FISH analyses
Metaphase spreads of HCT116 cells were obtained as described previously (Pantic et al., 2005). The number of analysed metaphases were between 9 and 34, depending on the proliferation capacity of the clones. Individual telomere length was analysed by Q-FISH as described recently (El-Daly et al., 2005). Telomere fluorescence intensity values were expressed in arbitrary units.
Multiplex (M)-FISH analysis
Combinatorial labeling of whole chromosome painting probes and hybridization were performed as described (Popp et al., 2000). For evaluation of hybridized metaphase spreads, a Leica DM RXA RF8 epifluorescence microscope (Leica Microsysteme, Bensheim, Germany) was used. Under control of the Leica Q-FISH software, images were acquired separately for each fluorochrome using a Sensys CCD camera (Photometrics, Tucson, AZ, USA) with a Kodak KAF 400 Chip. Images were processed using the Leica Multicolor Karyotyping (MCK) software package for spectral image analysis. Ten metaphase spreads were evaluated per cell line.
Data analysis was performed using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) and Origin 6.1 (Origin Lab Corporation, Northampton, MA, USA) software. Results are shown as means±s.e. (or s.d.) of values obtained in independent experiments. Student's t-test was used to determine statistical significance.
quantitative fluorescence in situ hybridization
telomere restriction fragment
fluorescence activated cell sorter
green fluorescent protein
fluorescence intensity of individual telomeres expressed in arbitrary units
phoenix ampho packaging cells
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We gratefully acknowledge the excellent technical assistance from I Skatulla. We also thank B Vogelstein for providing the HCT116 cells. In addition, we are indebted to R Weinberg and H Vaziri for providing the retroviral hTERT construct. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 364) (to UM), Deutsche Krebshilfe eV (to PB) and from the European Union (QLG1-CT-1999-01341 and MOL CANCER MED) (to UM and PB).
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Pantic, M., Zimmermann, S., El Daly, H. et al. Telomere dysfunction and loss of p53 cooperate in defective mitotic segregation of chromosomes in cancer cells. Oncogene 25, 4413–4420 (2006). https://doi.org/10.1038/sj.onc.1209486
- telomere dysfunction
- telomerase inhibition
- chromosomal instability
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