Changes in the expression of telomere maintenance genes suggest global telomere dysfunction in B-chronic lymphocytic leukemia

Telomeres are nucleoprotein structures that cap chromosomes and shorten with each division. Telomere structure and functions depend on the telomerase enzyme (hTERT, hTR, DYSKERIN) for elongation,¹  on the shelterin complex (TRF1, TRF2, TIN2, hRAP1, TPP1, POT1) that regulates telomere length and protects them against degradation and fusion, and on a set of multifunctional factors, including RPA1, hEST1A, KU70/KU80 and the RAD50-MRE11-NBS1 complex²  (Figure 1A).

Telomerase activity is absent or very low in somatic cells and increased in proliferative lymphoid cells.³  In most cancer cells, the catalytic subunit of telomerase (hTERT) is overexpressed to allow their long-term proliferation.⁴  Research in oncogenesis is now focusing on the other telomeric genes, especially the shelterin complex.^5,,,,–10  Specific changes in the expression of these genes in cancers may provide new knowledge about oncogenesis and useful clinical markers, but would also lead to the development of new therapeutic agents.

B-cell chronic lymphocytic leukemia (B-CLL) results from the progressive accumulation of a leukemic clone (for review, see Chiorazzi and Ferrarini¹¹ ) that shows lower telomerase activity at disease onset¹²  and increased activity in advanced stages and bad prognosis group.¹³  Telomeres are shorter in B-CLL cells versus normal B cells, and especially short for patients with bad prognosis. Telomere length is thus a powerful prognostic marker for B-CLL.^13,14  In this work, we investigated whether the transcriptional status of the telomeric proteins is modified in B cells from B-CLL patients.

After consent was obtained in accordance with the Declaration of Helsinki and according to institutional guidelines, total blood samples were collected from 20 healthy donors (at the “Etablissement Français du Sang” of Lyon and Pitié-Salpétrière Hospital) and from 42 B-CLL patients (at the Lyon Sud and Pitié-Salpétrière Hospitals). Diagnoses were confirmed using morphology and flow-cytometry usual B-CLL characteristics (Matutes score ≥ 4). B lymphocytes were purified from peripheral blood by negative selection using the RosetteSep Human B-cell enrichment cocktail (Stem Cell Technologies, Vancouver, BC). The percentage of CD19⁺ cells was determined by cytometric assay using an α-CD19-PE antibody (GE Healthcare, Little Chalfont, United Kingdom). More than 75% and 90% of CD19⁺ labeling was obtained for normal B cells and B-CLL cells, respectively. Binet stage, karyotype, fluorescent in situ hybridization, and mutational status (analyses were carried out as previously described¹⁵ ) features are summarized in Table 1.

B cells cultured on 10% fetal bovine serum supplemented RPMI, under 5% CO₂ atmosphere, were treated 48 hours by 0.001% of SAC suspension (Staphylococcus aureus Cowan strain I; Calbiochem, San Diego, CA) and 1 ng/mL of interkeukin-2 (Boehringer, Reims, France). Telomerase activity was measured by TRAPeze assay following the kit instruction (Intergent, New York, NY).

RNA extracted from purified B cells (Nalgene, New York, NY) was reverse-transcribed using random hexamer, Superscript II, and dNTP (Invitrogen, Carlsbad, CA). Quantitative PCR (Epicentre, Invitrogen, QIAGEN, Courtaboeuf, France) on opticon 3 thermocycler (Bio-Rad, Hercules, CA) was runfollowing the producer instructions. Sequences of primers used for PCR are listed in Table 2.

Gene expression levels were normalized using 3 reference genes (RPL13A, β-ACTIN, and RPL19). Distribution and variance equality were analyzed for each gene, in normal and B-CLL populations. Three different tests were run: Student (for gaussian populations with equal variance), Welch (for gaussian populations with different variance), or Wilcoxon (for nongaussian populations with equal variance) to determine the P value.

We determined the mRNA level of telomeric proteins in B cells from 42 B-CLL patients and 20 healthy donors. As previously published, ZAP-70 and BCL2 expressions were increased and those of KI-67 and hTERT were decreased (respective ratios: 25.6, 21, 0.26, and 0.04)^12,16,–18  (Figure 1B,E). We further showed that levels of hTERT and telomerase activity are correlated in B cells from one patient and one healthy donor (Figure 1C,D). Both levels cannot be increased by mitogenic stimulation in the patient cells, in which no cycling activity was observed (data not shown). Among the other factors implicated in telomerase activity, the expression of DYSKERIN, POT1, and hEST1A is also significantly reduced (2.4-, 5.8-, and 4.8-fold, respectively), whereas the one of TPP1 is increased (5.1-fold). Concerning the other shelterin components, the mRNA levels are lower in B-CLL cells for TRF1 and hRAP1 (2.9- and 3.0-fold, respectively), slightly reduced for TRF2 (1.2-fold), and almost unchanged for TIN2. We also observed a decrease in mRNA level of KU80, MRE11, and RAD50 (2.1-, 2.4-, and 11.1-fold, respectively) (Figure 1B,E) and an increase in RPA1 (9.7-fold).

Although these changes have to be confirmed at the protein level, they are expected to greatly affect the function of telomeres in B-CLL cells. The lower expression level of various factors involved in telomerase activity should impair telomere regeneration at each S-phase. Moreover, the altered expression of telomere capping factors might disrupt the capping complex, facilitating telomere degradation and shortening independently of the telomerase status. This correlates with telomeric damages already observed in B-CLL cells.^11,19  Our results, together with the fact that these cells could proliferate at appreciable levels,²⁰  suggest that the short telomeres observed in B-CLL cells in comparison with normal B cells but also with other types of B-cell malignancies²¹  might result from specific defects in telomerase-dependent and telomerase-independent pathways of telomere elongation. This would also explain the shorter telomeres observed in patients with severe outcome, despite an increase in telomerase activity.¹³  Finally, our results suggest that B-CLL cells may be particularly sensitive to telomere-damaging drugs, such as BIBR32, which exerts a cytotoxic effect on B-CLL cells mainly by damaging telomeres.²² 

In conclusion, our results provide the first evidence of a global modification in the expression of telomeric genes in B-CLL, which is characterized by a low expression of many components involved in telomere elongation and capping.

The authors thank D.P.'s medical supervisor Pr Jean André and Corrine Béal for sampling.

La Ligue Nationale contre le Cancer supported E.G.'s laboratory (équipe labellisée); Institut Nationale du Cancer (program EPIPRO) supported E.G.'s and G.S.'s laboratories; and Association Recherche contre le Cancer (ARECA program on Epigenetic Profiling) financed C.T.R.'s fellowship and the laboratories of L.S. and J.D.

Contribution: D.P. performed research, interpreted data, and cowrote the article; C.T.R., A.B., A.R.C., and E.B.S. performed research; H.M.-B., E.C.-B., G.S., L.S., and J.D. collected biologic samples and reviewed the article; E.G. designed and coordinated the research, interpreted data, and cowrote the article.

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

Correspondence: Eric Gilson, UMR5239, Faculté de Médecine Lyon Sud, 165 Chemin du Grand Revoyet, 69495 Pierre Bénite, France; e-mail: eric.gilson@ens-lyon.fr.