B-cell chronic lymphocytic leukaemia (B-CLL), the most common haematological malignancy in Western countries (Hamblin, 2009), results from the progressive accumulation of a monoclonal CD5+ CD19+ B cell clone. This accumulation may result from a defect in cell death mechanisms. Several clinical studies suggest that telomere length may be a valuable prognostic factor for this disease (Ricca et al, 2007).
In vertebrates, chromosome extremities, the telomeres, consist of a repeated motif (TTAGGG) bound to six proteins, the shelterin. Loss of one of these proteins causes the telomeres to no longer be hidden from the DNA damage surveillance and chromosome ends become inappropriately processed by DNA repair pathways (Sfeir & de Lange, 2012).
The telomeres are replicated in germinal cells with the help of telomerase [for review see (Gilson & Geli, 2007)]. This enzyme is a reverse transcriptase composed by a catalytic subunit (TERT) and a RNA matrix (TERC). The telomerase activity is almost absent in somatic cells but is reactivated in nearly 85% of cancer cells (Hornsby, 2010). The remaining 15% of cancer cells maintain their telomere through a phenomenon called Alternative Lengthening of Telomere (ALT) (Cesare & Reddel, 2010). These cells harbour telomeres that are very heterogeneous in size as well as extra-chromosomal circular telomeric DNA repeats (ECTR). Among these ECTR, C-circles have been determined as being the most valuable and reproducible hallmark of the ALT phenomenon (Henson et al, 2009) and may be produced by histone chaperone ASF1 depletion (O'Sullivan et al, 2014).
Studies suggest that both telomerase down-regulation and changes in the composition of telomeric proteins are involved in the pathogenesis of B-CLL (Campbell et al, 2006; Poncet et al, 2008), a phenomenon that could take place very early in the disease onset (Augereau et al, 2011). Moreover, it has been shown that B-CLL cells resistant to DNA damage-induced apoptosis exhibit shorter overhang, an increase in the number of telomere dysfunction induced foci (TIF) and an accumulation of non-homologous end-joining DNA repair system proteins (Brugat et al, 2010a,b). Lin et al (2010) also showed that telomere length could be correlated with disease stage, until reaching an extremely short size (beyond 1 kb) and undergoing fusion. This erosion is independent of the stage of the disease in ATM-mutated B-CLL (Britt-Compton et al, 2012). Finally, next generation sequencing identified somatic mutations in POT1 at low frequency (3·5% of all the cases analysed) that was correlated with telomere instability (Ramsay et al, 2013).
In vitro, the topoisomerase TOP3A disentangles migrating double Holliday junctions (dHJ) without leading to crossing over (Bussen et al, 2006). This process may occur in vivo with the cooperation of three other proteins: BLM, RMI1 and RMI2, known as the BTR (BLM/TOP3A/RMI) or dissolvasome complex (Singh et al, 2008). BLM and TOP3A are involved in the recombination process between telomeres characterized in ALT cells (Temime-Smaali et al, 2008). Hu et al (2001) also showed that the BLM/TOP3A complex is involved in the control of the sister chromatid exchange (SCE) level, which increases in BS cells, which are deficient in dissolvasome activity. This may be explained by the fact that dHJ are either dissolved by the dissolvasome complex without crossing over or resolved by GEN1 and/or the SLX4 associated nucleases MUS81 and SLX1A. In this case, crossing over could be induced (Wyatt et al, 2013). If dissolvasome is the preferred mechanism of dHJ disentanglement (Wechsler et al, 2011), dysfunction in dissolvasome activity could lead to an increase in crossing over frequency.
The present study aimed to determine the mode of telomere maintenance in B-CLL cells. We showed that neither telomerase nor ALT mechanisms are the main mechanisms for telomere maintenance in active circulating B cells. However we observed a high frequency of telomere sister chromatid exchange (T-SCE) in circulating B-CLL cells concomitantly with a down-regulation of TOP3A and with an increase in SLX1A, SLX4, MUS81 and GEN1 expression. Moreover, we showed a strong telomeric dysfunction in these cells, i.e., the down regulation of some shelterin components and an increased frequency of aberrant telomeric signals and TIF. Altogether, our results suggest that both telomere maintenance mechanisms, telomerase dependent and ALT, are dysfunctional in B-CLL. Furthermore, telomeric dysfunction, together with deregulation of the balance in dissolvasome/resolvasome in favour of the resolvasome, may account for the instability of telomeric sequence. This unbalanced dissolvasome/resolvasome may increase the telomere shuffling and help to manage the telomere erosion caused by the replication. Hence, targeting resolvasome activity with specific inhibitors may be relevant to treat this disease.
Material and methods
Patient samples, B-CLL purification
This research was approved by the Comité National d'Ethique de Recherche (Luxembourg, No 200509/05) and by the institutional review board protocol of the Hospices Civiles of Lyon (No 10-47). All participants gave written informed consents in accordance with the Declaration of Helsinki as revised in 2008. Clinical details, treatments and genomic characteristics of patients from the Luxembourg (Lu) cohort (N = 31, C1 to C31) and the Lyon (Ly) cohort (N = 20, C33 to C52) are summarized in Table 1. Peripheral blood samples were collected from patients and from healthy volunteers (Luxembourg: N = 10, V1 to V10, 28 to 55 years old, Lyon: N = 13, VL1 to VL13, 34 to 68 years old). Untreated B cells were purified by depletion using antibodies against CD2 and CD14 (MACS Microbeads; Miltenyi, Paris, France) Cell sorting purity was checked by cytometry analysis using a CD19 antibody (Becton Dickinson, Erembodegem, Belgium).
|C4||2003||III or IV||M||77||Leukeran, fludarabin, endoxan||Del 11q Del 13q||IGHV1-46*01 or *03 UM||Neg||Neg||98|
|C5||2005||I||M||70||No treatment||nd||IGHV4-61*02 M||Neg||Neg||nd|
|C6||2001||I||M||56||Fludarabin, endoxan||Del 13q Del 17p||nd||Neg||Pos||63|
|C8||2003||0–I||F||68||No treatment||Del 11q||IGHV3-33 UM||Neg||Pos||93|
|C11||2007||III||F||68||Leukeran||Del 13q Del 17p trisomy 12||UM||Pos||Pos||94|
|C14||1993||IV||M||82||No treatment||Del 13q||nd||Pos||Neg||96|
|C16||1991||I||F||72||No treatment||nd||IGHV1-3*01 M||nd||Neg||96|
|C24||1994||III||F||79||Mabthera||nd||IGHV3-30*03 or *18 M||Pos||Pos||81|
|C27||1985||IV||M||68||Leukeran, Fludarabin, Mabthera, Endoxan, Caelix, Aredia, Aranesp, Velcade||Del 11q Del 13q||nd||Neg||Neg||98|
|C28||2000||0||M||77||No treatment||nd||IGHV3-72*01 M||nd||nd||93|
|C31||1999||IV||M||68||Leukeran, Fludarabin, Endoxan, Mabthera||Normal||nd||Neg||Pos||98|
|C34||2012||III||M||74||No treatment||Del11q Del13q||nd||nd||nd||nd|
|C35||2001||IV||M||68||Rituximab, Bendumistin, Fludarabine||Del11q Del13q Del17p||nd||nd||nd||98|
|C36||2012||0||F||67||No treatment||Trisomy 12||UM||nd||Neg||75|
|C41||2010||I||F||70||No treatment||Del11q Del17p||nd||nd||Low||81|
|C45||2012||IV||M||80||No treatment||Del13q Trisomy 12||nd||nd||Neg||80|
|C49||2012||0||F||48||No treatment||Trisomy 12||UM||nd||Low||90|
|C50||2012||I||F||78||No treatment||Trisomy 12||M||nd||Pos||75|
|C51||2005||II||M||65||Rituximab, Fludarabine, Chloraminophen||Normal||nd||nd||nd||65|
We generated a TOP3A antibody in rabbit (Eurogentech, Liège, Belgium) using purified TOP3A according to the published protocol (Goulaouic et al, 1999). Primary antibodies against POT1 (EPR6319, Abcam, Cambridge, UK), TERF2 (4A794, Millipore, Erembodegem – Aalst, Belgium), 53BP1 (100-304, Novus, Woluwe-Saint-Lambert, Belgium), β-actin (A5441, Sigma, Pegasuslaan, Belgium), BrdU (B2531, Sigma) and rabbit anti mouse IgG (Z0259, Dako, Leuven, Belgium) were used.
cDNA synthesis and real time polymerase chain reaction (PCR)
Quantitative PCR and values normalization was performed according to Moussay et al (2010) using RNA18S1 as housekeeping gene. Primers and PCR conditions used are available on request. Single Tube TaqMan® Gene Expression Assays were ordered from Applied Biosystems (Life Technologies, Saint-Aubin, France) for the study of SLX1A (Hs02341353_g1), SLX4 (Hs00536164_m1), MUS81 (Hs01071851_g1) and GEN1 (Hs00416248_m1).
DNA was treated using the CpGenome Fast DNA Modification Kit (Millipore) according to the manufacturer's instructions and subjected to PCR. The PCR products were analysed by direct automatic PCR-assisted DNA dideoxynucleotide sequencing (Big Dye Terminator V3·1, Applied Biosystems, Halle, Belgium) on a 3130 Genetic Analyser (Applied Biosystems).
DNA constructs, SssI-induced methylation and luciferase assay
The TOP3A promoter region (−581 to −327) located upstream the minimal promoter and containing CpG islands found to be methylated in B-CLL patients was amplified by PCR from U2-OS genomic DNA and cloned upstream the CMV/EF1 promoter in the pCpGL-CMV/EF1 vector (Klug & Rehli, 2006) to generate the pCpGL-TOP3A (−581 to −327)-CMV/EF1 construct. Using the same approach, CpG islands mutations were introduced in this construct by cloning templates produced by DNA synthesis (Genewiz, London, UK). SssI-induced CpG island methylation in pCpGL-TOP3A (−581 to (−327)-CMV/EF1 constructs was performed with SssI methyltransferase (NEB, Leiden, The Netherlands) according to the manufacturer's instructions. Cells were harvested 24 h after transfection and assayed for luciferase and β-galactosidase activities using the pCH110 β-galactosidase plasmid as internal control.
Telomere length estimation
Telomere Restriction Fragment (TRF) of six cell lines was estimated by Southern blot according to Gomez et al (2006) and compared with the relative telomere length (RTL) determined by flow-fluorescence in situ hybridization experiments using the Telomere PNA Kit/FITC for Flow Cytometry (Dako) following the manufacturer's instruction. Data were recorded using a BD FACSCanto II (Becton Dickinson) with a collection stop on 20,000 single cells in G1 phase of the cell cycle.
The ploidy of each sample cell line, patients and controls, was estimated using propidium iodide (PI),with chicken red blood cells and 1301 cells as internal controls after fixation with ethanol and RNase treatment.
RTL was estimated as fluorescence intensity relative to the 1301 cells after ploidy correction. RTL = [(mean FL1 sample cells with probe – mean FL1 sample cells without probe) × DNA index of control cells × 100)/((mean FL1 control cells with probe – mean FL1 control cells without probe) × DNA index of sample cells] in %.
Rolling circle amplification of C-circle was performed as described (Gil & Coetzer, 2004; Lau et al, 2013). Briefly, 16 ng of genomic DNA were incubated for 17 h at 30°C with 3·75 units of φi29 DNA polymerase (NEB), before heat-inactivation (65°C for 10 min). For each condition, a control reaction without φ29 was run (φ−). Telo-PCR were run on the 480 Light Cycler (Roche, Houwald, Luxembourg), in triplicate and 36B4-PCR in duplicate, using the LightCycler® FastStart DNA Master SYBR Green I (Roche). Telomere and 36B4 initial concentrations [(Telo) and (36B4)] were calculated (in ng/μl) from to their respective standard curves. Results correspond to the following calculation: [(Telo) φ + /(36B4) φ + ]−[(Telo) φ-/(36B4) φ-].
Immunofluorescence experiments were performed on cytospin slides according to Chebel et al (2009).
Metaphase spreads, BrdU incorporation, T-SCE
Cells were cultured for 48–72 h in complete RPMI 1640 medium complemented with Interleukin-2/DSP30 mix according to the manufacturer's instructions (Premix; Amplitech, Compiègne, France). After mitotic arrest (Colcemid 0·4 μg/ml, 30 min, 37°C) and hypotonic treatment (0·075 mol/l KCl, 40 min, 37°C), cells were fixed in methanol/acetic acid (3:1) before being spread on slides. BrdU incorporation was checked with the mouse Apaap monoclonal antibody (D0651, Dako) and with the fast red substrate system (K0699, Dako) according to manufacturer's instructions. T-SCE rates were evaluated according to Tilman et al (2009) on B cells as well as in parallel on U2-OS and Hela cell lines as positive and negative controls.
Statistical analysis was carried out using a Wilcoxon rank-sum test (Mann–Whitney two tailed) of the GenStat Release 14·2 software (VSN International, Hemel Hempstead, UK). P values <0·05 were considered statistically significant.
Telomerase and ALT mechanisms are mainly absent in circulating B-CLL cells
In order to study the implication of the telomerase-dependent and independent telomere maintenance mechanism in our B-CLL patients, we first measured the expression of the telomerase enzyme (TERT) (Fig 1A) and found that TERT expression is very low in healthy volunteers and even lower in B-CLL patients, as already suggested by Poncet et al (2008).
As ALT cells harbour longer and more heterogeneous telomeres than telomerase-positive cells and in order to determine the telomere maintenance mechanism occurring at B-CLL telomeres, we first evaluated the length of telomeres of 31 B-CLL patients by non-radioactive flow cytometry (Fig 1B). Two different populations of B-CLL cells were identified: a major one with telomeres of 3 to 6 kb and a minor one with telomeres of 8 to 10 kb. As cells maintaining their telomeres with the ALT mechanism possess telomeres ranging from 2 to > 50 kb with a mean of 20 kb within an individual cell (Henson et al, 2002), no B-CLL patient can maintain their telomeres with an ALT mechanism. In order to confirm this hypothesis, we measured the concentration of C-circle, an ECTR, the most significant hallmark of the ALT activity (Henson et al, 2009). The C-circle concentration is slightly increased in B-CLL compared to the concentration measured in normal B cells (Fig 1C). However, this concentration is much lower than the C-circle concentration observed in the U-2 OS ALT cell line and remains at the same range as that observed in telomerase positive cell line (Fig 1C). Altogether these results suggest that telomerase and ALT activities were mainly absent in B-CLL patient's cells.
B-CLL patients present a high T-SCE rate
In order to definitively exclude the presence of an ALT activity in B-CLL patients, we evaluated the T-SCE rate, which is known to be increased in ALT cells. We stimulated cells from 3 newly recruited healthy volunteers as well as 12 newly recruited B-CLL patients. In order to remove false positives coming from BrdU misincorporation, we evaluated the incorporation of BrdU on the metaphase spreads by immuno-chemistry (Fig 1D, middle panel). Three kinds of metaphases have been observed (Fig 1D, top panel). Only patients with more than 2/3 BrdU positive metaphases were considered. As shown in Fig 1D (bottom panel), in healthy B cells, T-SCE occurred in about 30% of chromosomes extremities.
Hence in normal B cells, the exchange rate was already higher than that observed in our ALT-positive U-2 OS cell line used as control (an average of 3 to 10 exchanges per metaphase) (not shown). In B-CLL cells, chromatin was exchanged between nearly all the extremities as about 80% of extremities showed T-SCE (Fig 1D, bottom panel). This suggested that the telomere maintenance mechanism taking place in B-CLL cells was not identical to the already described ALT mechanism.
Altogether these results suggested that telomeres could be maintained through a recombinational mechanism different from that already described for ALT.
TOP3A is down-regulated in B-CLL
In order to explain the high T-SCE frequency observed in B-CLL we focused our work on factors that could influence this recombination rate. It has been previously shown that WRN and BLM are involved in T-SCE mechanism in ALT cells (Mendez-Bermudez et al, 2012). Hence we evaluated, in B-CLL patients and healthy volunteers, the transcription level of BLM and WRN, as well as TOP3A, the principal partner of BLM and the other known helicase partners of TOP3A, RECQL and RECQL5 (Fig 2A). The gene coding for TOP3A was strongly down regulated in both cohorts of patients, but not the genes coding for BLM and the other RecQ helicases we tested. In order to confirm that TOP3A was down regulated at protein level, we performed Western blot experiments on seven healthy volunteers and 19 B-CLL freshly recruited patients. TOP3A is also strongly down regulated at the protein level in B-CLL patients (Fig 2B). To explain this down-regulation, we generated the methylation map of the TOP3A promoter and found nine methylated CpG in the 5′ part of the promoter in B-CLL patients (Fig 2C). To precisely determine their implication, we used a luciferase based-approach in combination with SSSI-induced methylation and found that this epigenetic modification can influence TOP3A expression but not through CpGs found to be methylated in B-CLL (Fig 2D). Therefore, we conclude that TOP3A is not regulated through methylation of CpG islands in the TOP3A promoter in B-CLL.
Altogether, these data suggested that dissolvasome activity might be impaired in B-CLL patients through the down regulation of TOP3A. This impaired activity may account for the increase in T-SCE frequency characterized in B-CLL patients.
TOP3A down regulation could be involved in the telomere dysfunction observed in B-CLL
As down regulation of TOP3A by siRNA led to an increase in the number of TIF (Temime-Smaali et al, 2008) and as TOP3A protein level is low in our B-CLL patients compared to normal B cells, we evaluated the number of TIF in B-CLL. TIF were strongly increased in B-CLL cells, from 5% to 15%, as compared to the controls (Fig 3A). However, if considering only B-CLL, no clear correlation was observed between the TOP3A expression level and occurrence of TIF (Fig 3A), suggesting that TOP3A down regulation may not be the only factor involved in TIF occurrence.
Inhibition of TOP3A expression has been shown to lead to an increase in anaphase bridges number (Temime-Smaali et al, 2008). We therefore evaluated the telomeric dysfunction through the occurrence of abnormal telomeric signal in metaphase spreads obtained during our T-SCE experiments. The number of sister chromatids fusion, signal free ends, multiple telomeric signals and fusions are all increased in B-CLL telomeres as compared to those obtained from normal B cells (Fig 3B). These data support the hypothesis of TOP3A involvement of in telomere dysfunction in B-CLL. However, other factors known to impact the telomere maintenance had to be analysed.
Shelterin components are dysfunctional in B-CLL
In B-CLL, TIF frequency has been correlated with the down regulation of 2 shelterin genes, TPP1 and TINF2 (TIN2) (Augereau et al, 2011); therefore we evaluated the transcription levels of all shelterin genes. TERF1 (TRF1), TERF2 (TRF2) and POT1 expression were down regulated and differences were statistically significant for TERF1 and TERF2 (Fig 4A). These results recapitulate previous finding (Poncet et al, 2008
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