Cells of B-cell chronic lymphocytic leukemia (B-CLL) are characterized by short telomeres despite a low proliferative index. Because telomere length has been reported to be a valuable prognosis criteria, there is a great interest in a deep understanding of the origin and consequences of telomere dysfunction in this pathology. Cases of chromosome fusion involving extremely short telomeres have been reported at advanced stage. In the present study, we address the question of the existence of early telomere dysfunction during the B-CLL time course. In a series restricted to 23 newly diagnosed Binet stage A CLL patients compared with 12 healthy donors, we found a significant increase in recruitment of DNA-damage factors to telomeres showing telomere dysfunction in the early stage of the disease. Remarkably, the presence of dysfunctional telomeres did not correlate with telomere shortening or chromatin marks deregulation but with a down-regulation of 2 shelterin genes: ACD (coding for TPP1; P = .0464) and TINF2 (coding for TIN2; P = .0177). We propose that telomeric deprotection in the early step of CLL is not merely the consequence of telomere shortening but also of shelterin alteration.

Telomere erosion in proliferating cells leads to senescence1  and is thereby a potent barrier for tumor progression.2  In normal proliferating cells, such as germ and stem cells and in cancer cells, this erosion is compensated mainly by a specialized reverse transcriptase named telomerase.3,4  This enzyme is crucial in cancer cells for tumor progression.5  However, telomere shortening because of telomerase invalidation can also cooperate with p53 deficiency to facilitate carcinogenesis in aged mice.6  In chronic lymphocytic leukemia (CLL), despite a low proliferative index, telomeres are abnormally shortened7  and we previously reported a low telomerase expression level.8  In this context, both the consequence and the origins of telomere shortening are still a matter of debate.9  This point is of great importance as telomere shortening has been shown to be an even better prognosis marker than the reference one, namely the mutational status of IGHV gene.10  In this way, recent publications reported the presence of deprotected telomeres in radio-resistant patient cells11  and fused telomeres bearing extremely short telomeres in patients mainly at advances stages of the disease.12  Both of these studies proposed that telomere dysfunction or crisis can be at the origin of genomic instability and karyotype abnormality accumulation observed during disease progression.

A priori, the telomere injuries observed in CLL could stem from an excessive telomere shortening or alteration in the protective chromatin structure that cap telomeres or both. Indeed, if maintenance of telomere length depends on telomerase,13  telomere protection involves various telomere-associated proteins, such as the shelterin complex1  composed of 6 proteins (TRF1, TRF2, RAP1, TIN2, TPP1, POT1)14  (Figure 1). This complex appears to act as a hub that protects telomeres from being recognized as a double-strand break, thereby avoiding inappropriate DNA damage response (DDR) activation and aberrant repair15  (Figure 1). This complex also controls telomere length, by regulating both the telomerase activity16-19  and the alternative lengthening of telomere (ALT) mechanism.20  These proteins are also involved in oncogenesis. For instance, TRF2 is up-regulated in various types of human tumors21  and has been found to display oncogenic properties in mouse keratinocytes22  and human cancer cell lines.23  Human telomeres are also composed of nucleosomes that exhibit repressive chromatin marks, such as H3K9 and H4K20 trimethylation and low histone acetylation level24  associated to a hypermethylation of CpG in the subtelomeric regions. In agreement with a role for these chromatin modifications in telomere length control, the invalidation of enzymes responsible for their formation, that is, histone methyltransferase such as Suv39h1, Suv39h2, Suv4-20h, or DNA methyl transferase as DMNT1, 3a and 3b, leads to changes in telomere size.25-27 

Figure 1

The shelterin complex. Telomere DNA extremity is schematized at the junction between double strand DNA an 3′ single-strand overhang. The shelterin complex is composed of 2 double-strand telomeric sequence–binding proteins (TRF1 and TRF2), of one 3′overhang-binding protein (POT1) and of 3 others proteins (TIN2, TPP1, RAP1), essential for the complex constitution and stabilization. This complex inhibits DDR activation and ATM and/or ATR activation.

Figure 1

The shelterin complex. Telomere DNA extremity is schematized at the junction between double strand DNA an 3′ single-strand overhang. The shelterin complex is composed of 2 double-strand telomeric sequence–binding proteins (TRF1 and TRF2), of one 3′overhang-binding protein (POT1) and of 3 others proteins (TIN2, TPP1, RAP1), essential for the complex constitution and stabilization. This complex inhibits DDR activation and ATM and/or ATR activation.

Close modal

We have previously shown a global deregulation of the expression of shelterin proteins in an heterogeneous cohort of CLL patients.8  Here, we address the question of whether telomere dysfunction occurs at early stages of CLL and, if yes, whether it can be correlated with telomere shortening and/or changes in the telomere chromatin cap. As an early sign of telomere dysfunction, we monitored the recruitment at telomeres of DDR proteins. Thus, we restricted our analysis to B cells from 23 patients of first stage of CLL leukemogenesis (Binet stage A, < 2 years of disease history, previously untreated). We used B cells from 12 healthy age-matched donors as control.

We found a significant increase in telomeric damage in CLL patients in comparison with healthy donors. Strikingly, the presence of dysfunctional telomere at this early stage of the disease did not correlate with short telomeres but rather with a down-regulation of 2 shelterin genes: ACD and TINF2. We thus propose that early telomere deprotection in CLL results from shelterin dysregulation rather than from telomere shortening.

Patients and healthy donors

After informed consent and according to institutional guidelines, total blood samples were collected from 23 CLL patients at the Hospices Civils de Lyon and Grenoble University and from 12 age-matched healthy donors at the Etablissement Français du Sang. The protocol has been approved by the institutional review board. The diagnosis was based on morphologic criteria and immunophenotyping (Matutes score > 3/5). Patients were newly diagnosed (< 2 years of disease history) stage A patients, not previously treated by chemotherapy. Disease stage, FISH data, and IGH mutational status are provided for each patient in Table 1. Briefly, del13q, del11q, del17p, and t12 were, respectively, found in 56%, 8,7%, 4%, and 4% of patients and 22% of patients are unmutated. These percentages for some are inferior to what is found in general CLL patients,28  are in accordance with those observed in stage A patients.29,30 

Table 1

Sample characteristics

SampleDisease stageMutational statusKaryotypeFISH analysis
Available datas
17p1311q23del13q14+12CHIPIFqPCRTeloblot
CLL            
    ALD t + del m ✓ ✓ ✓ ✓ 
    ARM ✓ ✓ ✓ ✓ 
    BAR UM ND del ✓ ND ✓ ✓ 
    BRI ✓ ✓ ✓ ✓ 
    CAR ND del m ✓ ND ✓ ✓ 
    Dan37 del m ✓ ✓ ✓ ✓ 
    Dan39 del m ✓ ✓ ✓ ND 
    DUR ND del m + b ✓ ✓ ✓ ✓ 
    ECH ND del m ✓ ✓ ✓ ✓ 
    FRA UM ND ✓ ND ✓ ✓ 
    GUI ND ND ND ND 
    IRE UM del del m ✓ ND ND ✓ 
    JOS UM del ✓ ✓ ✓ ✓ 
    JOU ND del m + b ND ✓ ND ND 
    LOU ✓ ✓ ✓ ✓ 
    LUC A (complex) ✓ ✓ ✓ ✓ 
    MAR ND del b ✓ ✓ ✓ ✓ 
    MAR38 A (+12) +12 ✓ ✓ ✓ ✓ 
    MAR6 del m + b ✓ ✓ ✓ ✓ 
    PIE del m + b ✓ ✓ ✓ ✓ 
    RIC ✓ ✓ ✓ ✓ 
    SOL UM del m ✓ ✓ ✓ ✓ 
    SUZ del m ✓ ✓ ✓ ✓ 
Co            
    N120 NA NA NA NA NA NA NA ✓ ✓ ND ✓ 
    N121 NA NA NA NA NA NA NA ✓ ✓ ✓ ✓ 
    N122 NA NA NA NA NA NA NA ✓ ✓ ND ✓ 
    N123 NA NA NA NA NA NA NA ✓ ✓ ND ✓ 
    N124 NA NA NA NA NA NA NA ✓ ✓ ✓ ✓ 
    N125 NA NA NA NA NA NA NA ✓ ✓ ✓ ✓ 
    N126 NA NA NA NA NA NA NA ✓ ✓ ✓ ✓ 
    N127 NA NA NA NA NA NA NA ✓ ND ND ✓ 
    N128 NA NA NA NA NA NA NA ✓ ✓ ✓ ✓ 
    N129 NA NA NA NA NA NA NA ✓ ✓ ND ✓ 
    N132 NA NA NA NA NA NA NA ✓ ✓ ✓ ✓ 
    N133 NA NA NA NA NA NA NA ✓ ✓ ND ✓ 
SampleDisease stageMutational statusKaryotypeFISH analysis
Available datas
17p1311q23del13q14+12CHIPIFqPCRTeloblot
CLL            
    ALD t + del m ✓ ✓ ✓ ✓ 
    ARM ✓ ✓ ✓ ✓ 
    BAR UM ND del ✓ ND ✓ ✓ 
    BRI ✓ ✓ ✓ ✓ 
    CAR ND del m ✓ ND ✓ ✓ 
    Dan37 del m ✓ ✓ ✓ ✓ 
    Dan39 del m ✓ ✓ ✓ ND 
    DUR ND del m + b ✓ ✓ ✓ ✓ 
    ECH ND del m ✓ ✓ ✓ ✓ 
    FRA UM ND ✓ ND ✓ ✓ 
    GUI ND ND ND ND 
    IRE UM del del m ✓ ND ND ✓ 
    JOS UM del ✓ ✓ ✓ ✓ 
    JOU ND del m + b ND ✓ ND ND 
    LOU ✓ ✓ ✓ ✓ 
    LUC A (complex) ✓ ✓ ✓ ✓ 
    MAR ND del b ✓ ✓ ✓ ✓ 
    MAR38 A (+12) +12 ✓ ✓ ✓ ✓ 
    MAR6 del m + b ✓ ✓ ✓ ✓ 
    PIE del m + b ✓ ✓ ✓ ✓ 
    RIC ✓ ✓ ✓ ✓ 
    SOL UM del m ✓ ✓ ✓ ✓ 
    SUZ del m ✓ ✓ ✓ ✓ 
Co            
    N120 NA NA NA NA NA NA NA ✓ ✓ ND ✓ 
    N121 NA NA NA NA NA NA NA ✓ ✓ ✓ ✓ 
    N122 NA NA NA NA NA NA NA ✓ ✓ ND ✓ 
    N123 NA NA NA NA NA NA NA ✓ ✓ ND ✓ 
    N124 NA NA NA NA NA NA NA ✓ ✓ ✓ ✓ 
    N125 NA NA NA NA NA NA NA ✓ ✓ ✓ ✓ 
    N126 NA NA NA NA NA NA NA ✓ ✓ ✓ ✓ 
    N127 NA NA NA NA NA NA NA ✓ ND ND ✓ 
    N128 NA NA NA NA NA NA NA ✓ ✓ ✓ ✓ 
    N129 NA NA NA NA NA NA NA ✓ ✓ ND ✓ 
    N132 NA NA NA NA NA NA NA ✓ ✓ ✓ ✓ 
    N133 NA NA NA NA NA NA NA ✓ ✓ ND ✓ 

NA indicates nonapplicable; ND, not done; M, mutated; UM, unmutated; A, abnormal; N, normal; del, deletion; m, monoallelic; b, biallelic; del, deletion; +12, trisomy 12; and t, translocation.

Isolation of human B cells

B lymphocytes were purified from peripheral blood by using the human CD19 Microbeads kit (Miltenyi Biotec). Briefly, Ficoll prepared PBMCs were incubated with a cocktail of anti-CD19 antibody-conjugated magnetic microbeads. The magnetically labeled CD19-positive cells are collected by passing over a MACS column in a magnetic MACS separator. Cells were washed 3 times, eluted from the column and tested for CD19 enrichment. Percentage of CD19-positive cells was determined by flow cytometry (LSRII; BD Biosciences Pharmingen) using an α-CD19–PE antibody (Amersham Pharmacia Biotech). Over 93% of CD19-positive labeling was obtained for normal B cells and CLL cells. For CHIP experiments only, patient blood samples showing > 80% B-CLL cells were not further purified after Ficoll gradient separation.

Immunofluorescence

Cells were fixed in cold methanol (−20°C), washed twice in PBS, incubated 40 minutes in 1× Target Retrieval Solution (DAKO) at 95°C, cooled 20 minutes at room temperature, and rinsed 3 times in distilled water. Slides were then incubated 1 hour in blocking solution (50mM NaCl, 0.5% Triton X-100, 3% dry skimmed milk in PBS) and immunostained overnight at 37°C by 1:300 rabbit anti-53BP1 (NB 100-305; Novus Biologicals) and mouse anti-TRF1 (ab10579; Abcam) in blocking solution. After 3 rinses in 0.8%, PBS, 1.5% dry skimmed milk, 50% NaCl, slides were incubated 1 hour in blocking solution containing Alexa Fluor 555–conjugated anti–mouse (A-31 570; Molecular Probes/Interchim) and Alexa Fluor 488 anti–rabbit (A-21 206; Molecular Probes) antibodies at 37°C. Slides were mounted in VectaShield antifade reagent (Vector Laboratories/CliniSciences) and observed thanks to a confucal microscope (Axioplan2 LSM510, Carl Zeiss) with plan achromal 63×/1.4 oil objective and the Zeiss LSM510 v3.2 acquistion software. Optical sections were recorded at room temperature with a z-step < 200 nm to determine the occurrence of 53BP1/TRF1 colocalizations, at least 30 nuclei per condition were analyzed.

Chromatin immunoprecipitation

Formaldehyde-fixed cells (0.75%) were washed and lysed. After nuclei membrane lysis, chromatin was recovered and sonicated on BioRuptor Sonicator (Diagenode). Chromatin (about 800-bp fragments) was separated in different fractions (corresponding to 2.5 million cells for the IgG and 1 million cells for the input and each histone directed antibody used), and precleared (premixed protein A and G sepharose 4 Fast-Flow 50%/50%; Amersham). Fractions were incubated overnight at 4°C with 5 μg of anti H3K9 trimetlyated (07-442; Milipore), H3K9 acetylated (07-352; Milipore), total H3 (ab1791; Abcam), or 10 μg of irrelevant mouse immunoglobulin (I8765; Sigma-Aldrich). After 1 hour of incubation premixed beads were washed 4 times in buffer of increase stringency. Eluates were, subjected to RNAse A treatment (1 hour, 37°C, 1 mg of RNAse A; Roche), reverse cross-linked (65°C, overnight in 20mM NaCl). Protein digestion was done at 45°C for 1 hour with 20 mg of Proteinase K (Roche). DNA was purified (classic phenol/chloroform method) before being spotted thanks to Bio-Dot SF system (Bio-Rad). Ten percent of DNA precipitated was spotted on a Hybon N+ membrane (GE Healthcare). DIG-labeled (DIG-High Prime kit; Roche Applied Bioscience) telomeric (400 bp of repeated 5′-T2AG3-3′ motif) or ALU (173 bp of genomic DNA amplified using primers 5′-TGAAACCCCGTCTCTACTAAAA-3′ and 5′-GTCTCGCTCTGTCGCCCA-3′). (Plasmids are available on request to E.G. or B.H.) Probes were used for membrane hybridization. Chemiluminescent signals were scanned with a CDD camera image analyzer system (LAS-300; Fujifilm). Bands were quantified by Image J software. Signals were quantified thanks to a linear regression obtained by plotting the signal (arbitrary units) in function of the percentage of input.

Southern blot determination of telomere size (Teloblot)

Telomere size was determined by terminal repeat fragment (TRF) experiments, considering the maximal intensity peak by using Image J software. Briefly, genomic DNA was purified following the DNeasy blood and Tissue kit (QIAGEN) instructions. Four micrograms of DNA was digested overnight by 4 μL of HinfI and 4 μL of RsaI (New England Biolabs). After precipitation, DNA was diluted in 1× TE (Tris 10mM, EDTA 1mM) and subjected to 1% low-melting agarose (Bio-Rad) gel electrophoresis for 15 hours in Chef Mapper DRIII (Bio-Rad) at 3.5 V/cm2. DNA was blotted to N+ Hybond membrane (GE Healthcare) by capillarity transfer in SCC 20× buffer and then UV cross-linked. Hybridization and detection of the telomeric sequences were performed as for ChIP experiments.

RT and Q-PCR

RNA was extracted from 5 million purified B cells using the TRIzol reagent according to the manufacturer's instructions (Invitrogen) and reverse transcribed using the high-capacity cDNA reverse transcription kit (Applied Biosystems). Custom-made 384-well TaqMan Low Density Arrays (TLDA) were used for quantitative PCR (Q-PCR) amplification with the TaqMan Gene expression master mix on a 7900HT Fast Real-Time PCR system (Applied Biosystems). The geometrical mean of 5 references genes was used for gene expression normalization. References genes (HDAC3, MBD4, WRN, DKC1, ATR) were selected by geNorm software (http://medgen.ugent.be/∼jvdesomp/genorm/) with a pairwise variation (V) inferior to 0.15.

Statistical analyses

Considering the sample size, we performed nonparametric 2-sample Wilcoxon rank-sum tests (also called the Mann-Whitney U test), using R software to test significance of mean differences in each experiment.

Study design

We performed our experiments on B lymphocytes purified from the peripheral blood of 12 healthy donors and 23 newly diagnosed and previously untreated stage A CLL patients coming from 2 different hospitals. The patients fulfilled the classic CLL phenotypic and molecular criteria (Table 1 and see “Patients and healthy donors”). As expected,31,32  the CLL patients exhibited an increased BCL2 expression and decreased Ki67 expression, known to be responsible for a higher antiapoptotic activity in a low proliferating rate context (Figure 2A). Importantly, this cohort of patients harbors the previously published features of telomere changes, that is, a general decrease in telomere length (P = .0025) in CLL lymphocytes compared with normal B cells and an even shorter length in IGVH unmutated versus mutated patients (P = .0015; Figure 2B). Therefore, we are studying a representative panel of mutated and unmutated patients as far as classic CLL markers and telomere length are concerned.

Figure 2

CLL classic molecular and telomeric features. (A) BCL2, MKI67, and CDKN2A (respectively coding for Bcl2, Ki67, and p16) normalized gene expression level, quantified in purified B cells from 6 healthy donors and 20 stage A CLL patients. Wilcoxon test P values are indicated in each corresponding box plot. (B) Telomeric size was analyzed by Teloblotting on B lymphocytes purified from 12 healthy donors and 20 CLL patients. Box plot showing telomere size, with corresponding Wilcoxon test calculated P values in healthy (Co) versus CLL (CLL) B cells (left) and in mutated (M) versus unmutated (UM) CLL B cells (right). A representative picture of telomeric restriction fragment analysis of one normal B cells (Co), 5 CLL mutated patients (CL M), and 5 CLL unmutated patients (CLL UM) is shown (right). Telomere size (in kb) was calculated thanks to specific ladder, size is indicated on each side of the Teloblot.

Figure 2

CLL classic molecular and telomeric features. (A) BCL2, MKI67, and CDKN2A (respectively coding for Bcl2, Ki67, and p16) normalized gene expression level, quantified in purified B cells from 6 healthy donors and 20 stage A CLL patients. Wilcoxon test P values are indicated in each corresponding box plot. (B) Telomeric size was analyzed by Teloblotting on B lymphocytes purified from 12 healthy donors and 20 CLL patients. Box plot showing telomere size, with corresponding Wilcoxon test calculated P values in healthy (Co) versus CLL (CLL) B cells (left) and in mutated (M) versus unmutated (UM) CLL B cells (right). A representative picture of telomeric restriction fragment analysis of one normal B cells (Co), 5 CLL mutated patients (CL M), and 5 CLL unmutated patients (CLL UM) is shown (right). Telomere size (in kb) was calculated thanks to specific ladder, size is indicated on each side of the Teloblot.

Close modal

Telomere dysfunction in early stages of CLL oncogenesis

Recent studies8,10,33-35  have reported telomeric damage11,12  and imbalances of shelterin protein expression in CLL,8  but no study has as yet addressed the question of the origin of telomere damage in these patients. To determine whether CLL telomeres are deprotected and thereby recognized as DNA damages, we performed double immunofluorescence analyses: interphasic cells were immunostained with antibodies against TRF1 (a marker of telomeres) and 53BP1 (a DDR factor). The data were analyzed by confocal microscopy to score the number of 53BP1 foci colocalizing with telomeres forming the so-called TIFs (telomere dysfunction–induced foci36 ). In our experimental conditions, we only rarely detected the presence of 53BP1 foci in B cells from healthy donors (median of 0.03 focus per nucleus; Figure 3B-C). However, in CLL we found a significant 6-fold increase in 53BP1-positive foci (median of 0.18 focus per nucleus, P = .0485). As a comparison, we found a 17-fold increase in 53BP1 foci after 5Gy γ irradiation in one of the patients (Figure 3A). We observed an even more statistically relevant increase in TIF in CLL versus healthy donors (median of 0.08 vs 0 TIF per nucleus, P = .02352; Figure 3B-C). Thus, TIF represent roughly 44% of 53BP1 foci in B-CLL cells, thereby indicating that a significant proportion of DNA damage is linked to telomere dysfunction in CLL. In agreement with these median values, we were unable to detect TIF in 63% (7/11) of healthy and 22% (4/18) of CLL cells, allowing us to classify healthy and CLL donors in subgroups named TIF+ (presence of TIF) and TIF (absence of TIF).

Figure 3

Telomeric damage induced foci in B cells from healthy donors and CLL patients. Confocal immunofluorescence analyses of 53BP1 foci and TRF1/53BP1 colocalization (TIF). (A) CLL B cells isolated from one CLL patient were subjected (IR) or no (Co) to 5 Gy γ-IR and fixed for immunostaining 1 hour latter. Median number of 53BP1 foci and TIF per nucleus is shown. (B-C) B cells from 11 healthy donors (Co) and 18 CLL patients (CLL) were immunostained for 53BP1 and TRF1; 53BP foci and TIF were scored after confocal analysis. (B) Box plot showing number of 53BP1 foci (left) and TIF (right) per nucleus; Wilcoxon-calculated P values are indicated in each box plot. (C) Immunofluorescence representing pictures of 53BP1 foci (left), TRF1 labeling (middle), and TIF (right) in B cells from healthy donor (top) and CLL patient (bottom). Arrows indicate TIF; TOTO staining is outlined in the equatorial plane of the nucleus. Detailed of ×60 magnification are shown after z-stack reconstruction (Image J software).

Figure 3

Telomeric damage induced foci in B cells from healthy donors and CLL patients. Confocal immunofluorescence analyses of 53BP1 foci and TRF1/53BP1 colocalization (TIF). (A) CLL B cells isolated from one CLL patient were subjected (IR) or no (Co) to 5 Gy γ-IR and fixed for immunostaining 1 hour latter. Median number of 53BP1 foci and TIF per nucleus is shown. (B-C) B cells from 11 healthy donors (Co) and 18 CLL patients (CLL) were immunostained for 53BP1 and TRF1; 53BP foci and TIF were scored after confocal analysis. (B) Box plot showing number of 53BP1 foci (left) and TIF (right) per nucleus; Wilcoxon-calculated P values are indicated in each box plot. (C) Immunofluorescence representing pictures of 53BP1 foci (left), TRF1 labeling (middle), and TIF (right) in B cells from healthy donor (top) and CLL patient (bottom). Arrows indicate TIF; TOTO staining is outlined in the equatorial plane of the nucleus. Detailed of ×60 magnification are shown after z-stack reconstruction (Image J software).

Close modal

These data confirm a previous study showing the presence of TIF in CLL cells11  and further show that TIF occur during early stage of CLL. It is worth noting that the increase of TIF in CLL might have been underestimated because very short telomeres might not lead to detectable TRF1-positive foci.

Telomere deprotection in CLL patients does not correlate with mean telomere length

Because CLL cells have shorter telomeres than normal B cells, we asked whether the increased frequency of TIF in CLL patients is because of an excessive telomere shortening. Strikingly, whereas telomeres tend to be shorter in the TIF+ healthy subgroup, this is not the case in the TIF+ CLL subgroup (Figure 4). We also used as a cutoff the maximal number of TIF observed in healthy donors, this segregated CLL patients into 2 equal groups (TIF low and TIF high). Even using this method we failed to observed a diminution in telomere size in the TIF-high subgroup (supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Thus, if a shorter telomere length could be the principal cause of telomere dysfunction in normal B cells, it does not seem to be the case in CLL cells of stage A patients. This suggests that the appearance of TIF in early stages of the disease does not mainly result from replicative erosion but rather from altered capping functions. This does not exclude the possibility that some TIFs result from a complete or nearly complete deletion of telomeres as previously observed in more advanced stage.12  In agreement with this view, whereas no clear correlation is found between telomere shortening and TIF increase when normal individuals are compared with stage A patients (P = .1526), the correlation is significant (P = .0047) comparing normal individuals to 4 cases of stage B and C patients (supplemental Figure 1).

Figure 4

Telomere damages are not correlated with telomere shortening in CLL cells. Telomere restriction fragment analyzes were done on B lymphocytes purified from 12 healthy donors and 20 CLL patients. Box plot showing telomere size, with corresponding Wilcoxon test–calculated P values in cells bearing (TIF+) or not (TIF) TIF among the healthy (Co, left) or the CLL panel (CLL, right).

Figure 4

Telomere damages are not correlated with telomere shortening in CLL cells. Telomere restriction fragment analyzes were done on B lymphocytes purified from 12 healthy donors and 20 CLL patients. Box plot showing telomere size, with corresponding Wilcoxon test–calculated P values in cells bearing (TIF+) or not (TIF) TIF among the healthy (Co, left) or the CLL panel (CLL, right).

Close modal

Telomeric nucleosome status in CLL

Next, we wonder whether telomeric chromatin alterations could explain TIF formation in CLL cells. Thus, we performed ChIP experiments against histone modifications known to characterize “repressive” (H3 lysine 9 trimethylation: H3K9trim), or “active” chromatin (H3 lysine 9 acetylation: H3K9ac) and against total histone H3. We used an irrelevant IgG as a control and estimated telomeric versus Alu sequences enrichment in the immunoprecipitated fractions. We did not observe any significant alterations of the tested histone marks in CLL compared with normal samples, although we noted a trend toward an increased H3K9ac in CLL samples (P = .0539; Figure 5A-B). Interestingly, we also observed a propensity of the TIF+ healthy subgroup toward an increased presence of the H3K9ac mark at telomeres while this trend is totally lost in the TIF+ subgroups of CLL patients (Figure 5C). Although we failed to establish any statistically significant difference in the telomeric chromatin status of healthy versus CLL cells, our results are compatible with a common modification of chromatin state in CLL with a trend toward a more open conformation.

Figure 5

Telomeric histone modifications. (A-C) Immunoprecipitations against histone H3 (H3), trimethylated H3K9 (H3K9trim), and acetylated H3K9 (H3K9ac) were performed on chromatin isolated from B cells of 21 CLL patients and 12 healthy donors. An irrelevant IgG instead of specific antibodies was used in the control condition. Slot-blot analyses of telomeric and Alu sequences enrichment were performed using specific probes. Specific enrichment in immunoprecipitated fraction was estimated relatively to the INPUT fraction, and quantified thanks to a total input dilution range. (A,C) Box plots show relative enrichments of telomeric versus Alu sequences (T/A) for different immunoprecipitations. Corresponding Wilcoxon tests P values are inserted in box plots. (A) Comparison of ChIP realized on CLL B cells (CLL) versus normal B cells (Co) and (C) comparison of ChIP results obtained among normal (Co) or CLL (CLL) B cells bearing (TIF+) or not (TIF). (B) Representative result of a ChIP experiment performed on B cells from a CLL patient, showing, on the left the input range (IPT) used for the quantification and on the right the dots obtained after deposit of 10% of total H3, H3K9trim and H3K9ac immunoprecipitates. The membrane was revealed using ALU (top) or telomeric probes (bottom).

Figure 5

Telomeric histone modifications. (A-C) Immunoprecipitations against histone H3 (H3), trimethylated H3K9 (H3K9trim), and acetylated H3K9 (H3K9ac) were performed on chromatin isolated from B cells of 21 CLL patients and 12 healthy donors. An irrelevant IgG instead of specific antibodies was used in the control condition. Slot-blot analyses of telomeric and Alu sequences enrichment were performed using specific probes. Specific enrichment in immunoprecipitated fraction was estimated relatively to the INPUT fraction, and quantified thanks to a total input dilution range. (A,C) Box plots show relative enrichments of telomeric versus Alu sequences (T/A) for different immunoprecipitations. Corresponding Wilcoxon tests P values are inserted in box plots. (A) Comparison of ChIP realized on CLL B cells (CLL) versus normal B cells (Co) and (C) comparison of ChIP results obtained among normal (Co) or CLL (CLL) B cells bearing (TIF+) or not (TIF). (B) Representative result of a ChIP experiment performed on B cells from a CLL patient, showing, on the left the input range (IPT) used for the quantification and on the right the dots obtained after deposit of 10% of total H3, H3K9trim and H3K9ac immunoprecipitates. The membrane was revealed using ALU (top) or telomeric probes (bottom).

Close modal

A decreased expression of TINF2 and ACD correlates with TIF formation in CLL

Because loss or imbalance of shelterin components can induce TIF formation and because we previously reported a deregulation in shelterin gene expression in CLL, we asked whether TIF formation correlates with deregulation of shelterin expression in CLL cells. Therefore, we compared the mRNA expression levels of TERF1, TERF2, TINF2, TERF2IP (coding for RAP1), POT1, and ACD (coding for TPP1) between the TIF and TIF+ subgroup of CLL patients. There is a global tendency toward a down-regulation of shelterin component mRNA level in the TIF+ CLL subgroup (Figure 6). This is statistically significant for TINF2 (P = .0464) and ACD (P = .0177). This suggests that telomere dysfunction in CLL cells could result from an aberrant expression of TINF2 and ACD.

Figure 6

Shelterin component mRNA level in CLL B cells in function of TIF presence. Normalized genes expression level of TERF1, TERF2, RAP1, TINF2, ACD, and POT1. mRNA were extracted from purified B cells of 20 CLL patients demonstrating (TIF+) or not (TIF) telomeric damages. Wilcoxon test calculated P values are indicated in each corresponding box plot.

Figure 6

Shelterin component mRNA level in CLL B cells in function of TIF presence. Normalized genes expression level of TERF1, TERF2, RAP1, TINF2, ACD, and POT1. mRNA were extracted from purified B cells of 20 CLL patients demonstrating (TIF+) or not (TIF) telomeric damages. Wilcoxon test calculated P values are indicated in each corresponding box plot.

Close modal

We report here for the first time a telomere dysfunction in early stages of CLL. Interestingly, the presence of telomere DNA damage–induced foci (TIF) in CLL patients correlates with a reduced expression of the ACD and TINF2 shelterin genes but not with short telomeres.

It was previously reported that telomere shortening because of cell proliferation (in the absence of telomerase) results in TIF accumulation associated with an increase in telomeric H3K9 acetylation.37,38  In agreement with this scheme, we found that the presence of TIF in normal B cells tends to correlate with shorter telomeres and an increased presence of H3K9ac at telomere. However, there is no tendency toward telomere shortening in patients harboring a detectable level of TIF. Thus, the TIF increase in our cohort of stage A patients cannot be merely explained by telomere shortening because of cell overproliferation. In agreement with previous works showing an increased rate of proliferation39  and telomere crisis12  in advanced stage of CLL, we observed a significant correlation between the presence of TIF and short telomeres in patients at stages B and C. We therefore conclude that early stages of CLL is associated with telomere deprotection, which cannot be merely explained by telomere shortening, while advanced stages, probably as a consequence of an increased number of cell divisions, lead to critically short telomeres triggering DNA damage response and chromosome end fusion.

We noted a global decrease in the expression of shelterin component encoding genes in the stage A CLL patients harboring telomere damage and in particular a significant decrease in the expression of both ACD and TINF2 genes. Considering the shelterin complex conformation, TRF1 and TRF2 bind the duplex telomeric DNA and POT1 the single-strand 3′ overhang. POT1 function as an heterodimer with TPP1 (encoded by ACD) which enhances its binding to telomeres.12  TIN2 (encoded by TINF2) plays a major role in the complex stabilization, by bridging TPP1, TRF1, and TRF2.40,41  Thus, both ACD and TINF2 encode proteins playing a central role in the formation of the shelterin protein bridge linking the duplex part to the 3′ overhang of telomeric DNA. Their loss is thus expected to dissociate an essential capping structure.40-43  Therefore, we propose that increased TIF occurrence in stage A CLL patients results, at least in part, from a partial disassembly of the shelterin complex.

What could be the role of damaged telomeres in CLL pathogenesis? Because the presence of TIF is a hallmark of senescent cells36  and of senescent T lymphocytes in particular,44  we propose that early stage CLL is associated to accelerated B-lymphocyte senescence. This might result from the accumulation of various telomere dysfunctions in the B-cell lineage, including telomerase and shelterin down-regulation (8  and this work). In agreement with this view, CLL B cells are known to be initially G0 arrested45  and we observed an increase in p16 expression in our cohort of CLL patients (Figure 2A). Escape from senescence induce telomere-excessive shortening, fusion, and cell-crisis entry and promote genome instability.6  Increase in cell proliferation,39  fused-telomere and crisis-like phenotype have very recently been reported in more advanced stage of CLL.12  It could thus be possible that progression toward more advanced stage or transformation in the aggressive Richter syndrome are associated with senescence bypass.

To our knowledge, this work provides the first evidence of telomere deprotection in a human malignancy in link with an alteration of the shelterin complex. Interestingly, these damages occur at an early stage of the disease, suggesting that they contribute to the early step of malignant transformation. This enlightens the concept, well documented in mouse models but still hypothetical in human cancer, that telomere dysfunction facilitates cancer initiation.6,46 

This article is a continuation of a previous report.8 

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

The authors thank G. Belz for help on statistical analyses.

This work was supported by grants from la Ligue Nationale contre le Cancer (équipe labelisée), ARECA program on Epigenetic Profiling, INCa (program TELINCA and TELOFUN), and the European Union (FP7-Telomarker, Health-F2-2007-200950).

Contribution: A.A., C.T.d.R., T.S., S.B., B.H., and D.P. performed experiments; M.C., D.L., L.J., and G.S. collected biologic samples; and E.G. and D.P. designed the research, analyzed the results, made the figures, and wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Eric Gilson, University of Nice, Laboratory of Biology and Pathology of Genomes, UMR 6267 CNRS U998 Inserm, 28 avenue Valombrose, Faculté de Médecine 06107 Nice Cedex 2, France; e-mail: [email protected].

1
Gilson
 
E
Geli
 
V
How telomeres are replicated.
Nat Rev Mol Cell Biol
2007
, vol. 
8
 
10
(pg. 
825
-
838
)
2
Deng
 
Y
Chan
 
SS
Chang
 
S
Telomere dysfunction and tumour suppression: the senescence connection.
Nat Rev Cancer
2008
, vol. 
8
 
6
(pg. 
450
-
458
)
3
Greider
 
CW
Blackburn
 
EH
Identification of a specific telomere terminal transferase activity in tetrahymena extracts.
Cell
1985
, vol. 
43
 
2 Pt 1
(pg. 
405
-
413
)
4
Kim
 
NW
Piatyszek
 
MA
Prowse
 
KR
, et al. 
Specific association of human telomerase activity with immortal cells and cancer.
Science
1994
, vol. 
266
 
5193
(pg. 
2011
-
2015
)
5
Brunori
 
M
Luciano
 
P
Gilson
 
E
Geli
 
V
The telomerase cycle: normal and pathological aspects.
J Mol Med
2005
, vol. 
83
 
4
(pg. 
244
-
257
)
6
Artandi
 
SE
Chang
 
S
Lee
 
SL
, et al. 
Telomere dysfunction promotes non-reciprocal translocations and epithelial cancers in mice.
Nature
2000
, vol. 
406
 
6796
(pg. 
641
-
645
)
7
Rossi
 
D
Lobetti Bodoni
 
C
Genuardi
 
E
, et al. 
Telomere length is an independent predictor of survival, treatment requirement and Richter's syndrome transformation in chronic lymphocytic leukemia.
Leukemia
2009
, vol. 
23
 
6
(pg. 
1062
-
1072
)
8
Poncet
 
D
Belleville
 
A
t'kint de Roodenbeke
 
C
, et al. 
Changes in the expression of telomere maintenance genes suggest global telomere dysfunction in B-chronic lymphocytic leukemia.
Blood
2008
, vol. 
111
 
4
(pg. 
2388
-
2391
)
9
Jahrsdorfer
 
B
Weiner
 
GJ
Short telomeres in B-CLL: the chicken or the egg?
Blood
2008
, vol. 
111
 
12
pg. 
5756
  
author reply 5756–5757
10
Ricca
 
I
Rocci
 
A
Drandi
 
D
, et al. 
Telomere length identifies two different prognostic subgroups among VH-unmutated B-cell chronic lymphocytic leukemia patients.
Leukemia
2007
, vol. 
21
 
4
(pg. 
697
-
705
)
11
Brugat
 
T
Nguyen-Khac
 
F
Grelier
 
A
Merle-Beral
 
H
Delic
 
J
Telomere dysfunction-induced foci arise with the onset of telomeric deletions and complex chromosomal aberrations in resistant chronic lymphocytic leukemia cells.
Blood
2010
, vol. 
116
 
2
(pg. 
239
-
249
)
12
Lin
 
TT
Letsolo
 
BT
Jones
 
RE
, et al. 
Telomere dysfunction and fusion during the progression of chronic lymphocytic leukemia: evidence for a telomere crisis.
Blood
2010
, vol. 
116
 
11
(pg. 
1899
-
1907
)
13
Blackburn
 
EH
Greider
 
CW
Szostak
 
JW
Telomeres and telomerase: the path from maize, tetrahymena and yeast to human cancer and aging.
Nat Med
2006
, vol. 
12
 
10
(pg. 
1133
-
1138
)
14
de Lange
 
T
Shelterin: the protein complex that shapes and safeguards human telomeres.
Genes Dev
2005
, vol. 
19
 
18
(pg. 
2100
-
2110
)
15
Palm
 
W
de Lange
 
T
How shelterin protects mammalian telomeres.
Annu Rev Genet
2008
, vol. 
42
 (pg. 
301
-
334
)
16
Lei
 
M
Zaug
 
AJ
Podell
 
ER
Cech
 
TR
Switching human telomerase on and off with hPOT1 protein in vitro.
J Biol Chem
2005
, vol. 
280
 
21
(pg. 
20449
-
20456
)
17
Loayza
 
D
De Lange
 
T
POT1 as a terminal transducer of TRF1 telomere length control.
Nature
2003
, vol. 
423
 
6943
(pg. 
1013
-
1018
)
18
van Steensel
 
B
de Lange
 
T
Control of telomere length by the human telomeric protein TRF1.
Nature
1997
, vol. 
385
 
6618
(pg. 
740
-
743
)
19
Wang
 
F
Podell
 
ER
Zaug
 
AJ
, et al. 
The POT1-TPP1 telomere complex is a telomerase processivity factor.
Nature
2007
, vol. 
445
 
7127
(pg. 
506
-
510
)
20
Jiang
 
WQ
Zhong
 
ZH
Henson
 
JD
Reddel
 
RR
Identification of candidate alternative lengthening of telomeres genes by methionine restriction and RNA interference.
Oncogene
2007
, vol. 
26
 
32
(pg. 
4635
-
4647
)
21
Nakanishi
 
K
Kawai
 
T
Kumaki
 
F
, et al. 
Expression of mRNAs for telomeric repeat binding factor (TRF)-1 and TRF2 in atypical adenomatous hyperplasia and adenocarcinoma of the lung.
Clin Cancer Res
2003
, vol. 
9
 
3
(pg. 
1105
-
1111
)
22
Blanco
 
R
Munoz
 
P
Flores
 
JM
Klatt
 
P
Blasco
 
MA
Telomerase abrogation dramatically accelerates TRF2-induced epithelial carcinogenesis.
Genes Dev
2007
, vol. 
21
 
2
(pg. 
206
-
220
)
23
Biroccio
 
A
Rizzo
 
A
Elli
 
R
, et al. 
TRF2 inhibition triggers apoptosis and reduces tumourigenicity of human melanoma cells.
Eur J Cancer
2006
, vol. 
42
 
12
(pg. 
1881
-
1888
)
24
Blasco
 
MA
The epigenetic regulation of mammalian telomeres.
Nat Rev Genet
2007
, vol. 
8
 
4
(pg. 
299
-
309
)
25
Garcia-Cao
 
M
O'Sullivan
 
R
Peters
 
AH
Jenuwein
 
T
Blasco
 
MA
Epigenetic regulation of telomere length in mammalian cells by the Suv39h1 and Suv39h2 histone methyltransferases.
Nat Genet
2004
, vol. 
36
 
1
(pg. 
94
-
99
)
26
Benetti
 
R
Gonzalo
 
S
Jaco
 
I
, et al. 
Suv4-20h deficiency results in telomere elongation and derepression of telomere recombination.
J Cell Biol
2007
, vol. 
178
 
6
(pg. 
925
-
936
)
27
Gonzalo
 
S
Jaco
 
I
Fraga
 
MF
, et al. 
DNA methyltransferases control telomere length and telomere recombination in mammalian cells.
Nat Cell Biol
2006
, vol. 
8
 
4
(pg. 
416
-
424
)
28
Mehes
 
G
Chromosome abnormalities with prognostic impact in B-cell chronic lymphocytic leukemia.
Pathol Oncol Res
2005
, vol. 
11
 
4
(pg. 
205
-
210
)
29
Matthews
 
C
Catherwood
 
MA
Morris
 
TC
, et al. 
Serum TK levels in CLL identify Binet stage A patients within biologically defined prognostic subgroups most likely to undergo disease progression.
Eur J Haematol
2006
, vol. 
77
 
4
(pg. 
309
-
317
)
30
Rossi
 
D
Sozzi
 
E
Puma
 
A
, et al. 
The prognosis of clinical monoclonal B cell lymphocytosis differs from prognosis of Rai 0 chronic lymphocytic leukaemia and is recapitulated by biological risk factors.
Br J Haematol
2009
, vol. 
146
 
1
(pg. 
64
-
75
)
31
Hanada
 
M
Delia
 
D
Aiello
 
A
Stadtmauer
 
E
Reed
 
JC
bcl-2 gene hypomethylation and high-level expression in B-cell chronic lymphocytic leukemia.
Blood
1993
, vol. 
82
 
6
(pg. 
1820
-
1828
)
32
Klein
 
U
Tu
 
Y
Stolovitzky
 
GA
, et al. 
Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells.
J Exp Med
2001
, vol. 
194
 
11
(pg. 
1625
-
1638
)
33
Bechter
 
OE
Eisterer
 
W
Pall
 
G
Hilbe
 
W
Kuhr
 
T
Thaler
 
J
Telomere length and telomerase activity predict survival in patients with B cell chronic lymphocytic leukemia.
Cancer Res
1998
, vol. 
58
 
21
(pg. 
4918
-
4922
)
34
Grabowski
 
P
Hultdin
 
M
Karlsson
 
K
, et al. 
Telomere length as a prognostic parameter in chronic lymphocytic leukemia with special reference to VH gene mutation status.
Blood
2005
, vol. 
105
 
12
(pg. 
4807
-
4812
)
35
Roos
 
G
Krober
 
A
Grabowski
 
P
, et al. 
Short telomeres are associated with genetic complexity, high-risk genomic aberrations, and short survival in chronic lymphocytic leukemia.
Blood
2008
, vol. 
111
 
4
(pg. 
2246
-
2252
)
36
Takai
 
H
Smogorzewska
 
A
de Lange
 
T
DNA damage foci at dysfunctional telomeres.
Curr Biol
2003
, vol. 
13
 
17
(pg. 
1549
-
1556
)
37
d'Adda di Fagagna
 
F
Reaper
 
PM
Clay-Farrace
 
L
, et al. 
A DNA damage checkpoint response in telomere-initiated senescence.
Nature
2003
, vol. 
426
 
6963
(pg. 
194
-
198
)
38
Benetti
 
R
Garcia-Cao
 
M
Blasco
 
MA
Telomere length regulates the epigenetic status of mammalian telomeres and subtelomeres.
Nat Genet
2007
, vol. 
39
 
2
(pg. 
243
-
250
)
39
Messmer
 
BT
Messmer
 
D
Allen
 
SL
, et al. 
In vivo measurements document the dynamic cellular kinetics of chronic lymphocytic leukemia B cells.
J Clin Invest
2005
, vol. 
115
 
3
(pg. 
755
-
764
)
40
Ye
 
JZ
Donigian
 
JR
van Overbeek
 
M
, et al. 
TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on telomeres.
J Biol Chem
2004
, vol. 
279
 
45
(pg. 
47264
-
47271
)
41
O'Connor
 
MS
Safari
 
A
Xin
 
H
Liu
 
D
Songyang
 
Z
A critical role for TPP1 and TIN2 interaction in high-order telomeric complex assembly.
Proc Natl Acad Sci U S A
2006
, vol. 
103
 
32
(pg. 
11874
-
11879
)
42
Kim
 
SH
Beausejour
 
C
Davalos
 
AR
Kaminker
 
P
Heo
 
SJ
Campisi
 
J
TIN2 mediates functions of TRF2 at human telomeres.
J Biol Chem
2004
, vol. 
279
 
42
(pg. 
43799
-
43804
)
43
Tejera
 
AM
Stagno d'Alcontres
 
M
Thanasoula
 
M
, et al. 
TPP1 is required for TERT recruitment, telomere elongation during nuclear reprogramming, and normal skin development in mice.
Dev Cell
2010
, vol. 
18
 
5
(pg. 
775
-
789
)
44
Chebel
 
A
Bauwens
 
S
Gerland
 
LM
, et al. 
Telomere uncapping during in vitro T-lymphocyte senescence.
Aging Cell
2009
, vol. 
8
 
1
(pg. 
52
-
64
)
45
Zenz
 
T
Mertens
 
D
Kuppers
 
R
Dohner
 
H
Stilgenbauer
 
S
From pathogenesis to treatment of chronic lymphocytic leukaemia.
Nat Rev Cancer
2010
, vol. 
10
 
1
(pg. 
37
-
50
)
46
Rudolph
 
KL
Millard
 
M
Bosenberg
 
MW
DePinho
 
RA
Telomere dysfunction and evolution of intestinal carcinoma in mice and humans.
Nat Genet
2001
, vol. 
28
 
2
(pg. 
155
-
159
)

Author notes

*

A.A. and C.T.d.R. contributed equally to this study.

Sign in via your Institution