Supported by grants from Société Française du Cancer, Société Française d'Hématologie, Ligue Nationale contre le Cancer, Association pour la Recherche sur le Cancer and Commissariat à l'Energie Atomique.
Mutagenicity of non-homologous end joining DNA repair in a resistant subset of human chronic lymphocytic leukaemia B cells
Version of Record online: 20 MAR 2006
British Journal of Haematology
Volume 133, Issue 5, pages 520–525, June 2006
How to Cite
Deriano, L., Merle-Béral, H., Guipaud, O., Sabatier, L. and Delic, J. (2006), Mutagenicity of non-homologous end joining DNA repair in a resistant subset of human chronic lymphocytic leukaemia B cells. British Journal of Haematology, 133: 520–525. doi: 10.1111/j.1365-2141.2006.06071.x
- Issue online: 27 APR 2006
- Version of Record online: 20 MAR 2006
- Received 6 December 2005; accepted for publication 1 February 2006
- B-chronic lymphocytic leukaemia;
- non-homologous end joining DNA repair;
- genotoxic resistance
Non-homologous end joining (NHEJ) is an important determinant of genomic stability in mammalian cells. This DNA repair pathway is upregulated in a subset of B-cell chronic lymphocytic leukaemia (B-CLL) cells resistant to DNA damage-induced apoptosis. Using an in vitro assay for double-strand breaks (DSB) end ligation, we studied the fidelity of DSB repair in B-CLL cells which were resistant or sensitive to in vitro DSB-induced apoptosis with concomitant patients’ resistance or sensitivity to chemotherapy, respectively. The fidelity of DNA repair was determined by DNA sequencing of polymerase chain reaction products cloned in pGEM-T vector. Sequence analysis of DNA end junctions showed that the frequency of accurate ligation was higher in sensitive B-CLL cells and control cell lines, than in resistant cells where end joining was associated with extended deletions. Upregulated and error-prone NHEJ in resistant cells could be a quite possible mechanism underlying both genomic instability and poor clinical outcome.
Inaccurate repair or lack of repair of DNA double-strand breaks (DSB) can lead to apoptosis. Alternatively, they can lead to mutations or large-scale genomic instability through the generation of non-lethal chromosomal aberrations (Khanna & Jackson, 2001). There are two main pathways for DSB repair, homologous recombination (HR) and non-homologous end joining (NHEJ). NHEJ is the dominant mechanism for the repair of DSBs in mammalian cells. HR generally ensures error-free repair, but NHEJ can lead to loss or gain of a few nucleotides, as it rejoins the two DNA ends directly without search for homology. It requires the Ku70/Ku86 proteins which bind to DNA ends and recruit a protein kinase catalytic subunit (DNA-PKcs). Assembly of these proteins forms the DNA-PK complex, which, with XRCC4 and DNA ligase IV ligates broken ends (Khanna & Jackson, 2001). Several lines of evidence suggest that DNA-PK-proficient cells can display genomic rearrangements, in response to high doses of ionising radiation or after as few as two or three DSBs occurring simultaneously in the cell (Rothkamm et al, 2001). However, DNA-PK-dependent NHEJ protects against genome instability and NHEJ-deficient cells show higher rates of genome rearrangements, such as translocations (Difilippantonio et al, 2000; Ferguson et al, 2000). In addition to the classical NHEJ pathway, an alternative Ku-independent error-prone DSB repair pathway has been found in rodent and mammalian cells that involves microhomology-associated end joining (Feldmann et al, 2000; Bentley et al, 2004).
We recently reported that enhanced DNA-PK-dependent NHEJ inhibited DSB-induced apoptosis in some B-cell chronic lymphocytic leukaemia (B-CLL) cells (Deriano et al, 2005). These B-CLL cells, resistant to in vitro radiation-induced apoptosis, also present accelerated DNA repair kinetics after γ-irradiation and accumulate more chromosomal aberrations (reviewed by Guipaud et al, 2003). Using an in vitro assay for DSB end ligation, we studied the fidelity of the end-joining process in these resistant and sensitive B-CLL cells. There was decreased frequency of accurate ligation in resistant B-CLL cells. Furthermore, NHEJ proteins Ku70, DNA-PKcs and XRCC4 were required for end joining in both resistant and sensitive B-CLL cells.
Results and discussion
It was previously reported that some B-CLL patients presented B cells in which the NHEJ pathway was deregulated and consequently, these cells were resistant to DSB-induced apoptosis in vitro (Deriano et al, 2005). The present study analysed the accuracy of the DNA repair process in both, resistant and sensitive B-CLL cells. One B-CLL sample (U1) was sensitive to in vitro radiation-induced apoptosis. The second B-CLL sample (T2) was resistant to in vitro radiation-induced apoptosis. The clinical data for these two B-CLL patients are summarised in Table I.
|Patient no.||Sex/age*||Matutes score†||Year/stage‡||Year/treatment received||Year/lymphocyte count/μl||Response to treatment§||In vitro apoptosis (%): untreated/10 Gy γ-irradiation¶||P53||Karyotype|
|U1||F/58||5||1999/A 2003/B||1999/cyclosporine 2003/chloraminophene, corticoid||1999/50 000 2003/200 000 2004/15 000||Positive||12·5 ± 6/72 ± 10·5||Wild type||Trisomy 12|
|T2||M/79||5||1996/B 1999/B 2000/B 2003/B||1996/theophilline, chloraminophene 1999/CHOP, campath||1996/170 000 1999/200 000 2000/390 000 2003/300 000 2004/362 000||Negative||8·7 ± 4·4/10·5 ± 6·3||Mutated (220:Y C)||13q14.3 deletion p53 locus deleted|
In vivo, chemotherapy and radiation-induced DSBs are often chemically modified and comprise partially or completely incompatible DNA ends. We used substrates with compatible (BamHI) and incompatible (BamHI/PstI) DNA ends to study DNA repair fidelity (Fig 1A and B, upper panels). Overlapping junctions involving compatible DNA ends formed by the pairing of fortuitously complementary bases whereas accurate ligation of DNA ends with 5′ and 3′ single-stranded overhangs required fill-in DNA synthesis in a process by which DNA ends are transiently held together (Fig 1A and B, upper panels). Using extracts from U1 B-CLL cell, a control B-cell line and human glioma MO59K cells, NHEJ of incompatible (5′ and 3′ single-stranded overhangs) DNA ends was precise or associated with small single-stranded DNA deletions (incomplete fill in). In contrast, using T2 B-CLL cell extracts, end-joining products contained more deletion events (Fig 1A). Using T2 B-CLL extracts, NHEJ of compatible (5′ single-stranded overhangs) DNA ends also contained more deletion events than with U1 B-CLL cells, the B-cell line or MO59K cells (Fig 1B). During the end-ligation process, using either compatible or incompatible DNA substrates, T2 generated significantly more nucleotide deletion events than U1, the B-cell line and the MO59K cell line (P < 0·05 using the Fisher test) (Fig 1A and B). The error-prone joining observed with T2 B-CLL extract was not caused by high concentrations of Mg2+ in the extract as joining was inhibited by 10 mmol/l EDTA (Fig 1C).
The clones obtained for both types of DNA ends are described in Table II. The frequency of error-prone end-joining events was significantly higher for T2 than for each U1, the B-cell line and the MO59K cell line (P < 0·01, Fisher test). The average deletion sizes were 64 bp in T2, 23 bp in U1, 13 bp in the B-cell line and 8 bp in the MO59K cell line. The average deletion size in T2 was significantly larger than in U1, the B-cell line and the MO59K cell line (P < 0·01, t-test) and the number of end-joining events with the loss of more than 20 nucleotides was also significantly greater in T2 cells (P < 0·05, Fisher test) (Table II). According to previous reports, the majority of joins formed from a DSB by cell-free extracts from MO59K cells and lymphoid cells were accurate and produced by pairing of complementary bases or fill-in DNA synthesis (Bentley et al, 2004; Fig 1A and B, Table II). The average deletion size and the number of error-prone end-joining events in U1 were not significantly different from that in the B-cell line and the MO59K cell line. In contrast, NHEJ was error-prone and was associated with larger deletions in T2 B-CLL cells.
|Extract||Total clones*||Error-prone clones* (%)†||Microhomology (%)‡||Deletion >20 (%)‡||Deletion (bp) average|
|U1||54||23 (42·6)||15 (65·2)||4 (17·4)||23|
|T2||48||32 (66·7)§||18 (56·3)||15 (46·9)¶||64**|
|B cell line||43||17 (39·5)||10 (58·8)||1 (5·9)||13|
|MO59K||41||15 (36·6)||12 (80)||2 (13·3)||8|
Increased Ku-dependent NHEJ activity is associated with increased genomic instability in myeloid leukaemia (Gaymes et al, 2002) and G0 yeast cells with unrepressed NHEJ capacity have an increased frequency of small-scale mutations (Heidenreich et al, 2003). However, a recent study showed that error-prone DSB repair in high-grade human bladder cancer was not dependent on functional Ku70, DNA-PKcs or XRCC4 and involved microhomology-associated junctions (Bentley et al, 2004). We therefore tested whether T2 error-prone end joining is associated with such a process. Analysis of the products of NHEJ that had lost one or more nucleotides from the ends revealed two major classes of junctions: junctions with no homology and junctions at regions of microhomology (Table II). There was no significant difference between the frequency of microhomology at junctions in U1, T2, lymphoid and MO59K cells (P > 0·05, Fisher test). However, the ability to use microhomology could be restricted by the plasmid sequence itself and might not be representative of DNA variety in vivo. It has been recently reported that end joining becomes inefficient and more error prone in G0-arrested senescent cells; the ability of these cells to use microhomologies is also compromised (Seluanov et al, 2004). B-CLL is an age-related disease due to the accumulation of G0-arrested B cells. Malignant B cells with shorter telomeres (Damle et al, 2004), usually from patients with poor outcome, might have acquired such an aberrant end-joining process. Antibodies have been frequently used as specific reagents to test the involvement of candidate proteins in cell-free NHEJ. The inhibitor, NU7026, has been recently shown to inhibit specifically DNA-PKcs kinase activity and the DSB repair-associated activity (Deriano et al, 2005). Both NU7026 and anti-DNA-PKcs antibodies partially inhibited DNA end-joining reactions in U1, T2 and control cell lines (Fig 1D and E). The remaining end-joining products may be produced by DNA-PKcs-independent end joining. Moreover, both anti-Ku and anti-XRCC4 antibodies fully inhibited end joining in U1, T2, the B-cell line and the MO59K cell line (Fig 1E). Therefore, DNA-PK-dependent NHEJ can be error prone in some resistant B-CLL cells.
Together with our previous work (Vallat et al, 2003; reviewed by Guipaud et al, 2003), these results indicate that some B-CLL patients (that we defined as the resistant B-CLL subset) can present malignant B cells with: (i) higher than normal DNA-PKcs activity leading to resistance to genotoxic treatments; (ii) upregulated DNA-PK-dependent NHEJ activity leading to resistance to DSB-induced apoptosis; (iii) an increased number of chromosomal aberrations; and (iv) an error-prone DNA end-joining pathway, potentially leading to genomic instability and to more severe disease.
Indeed, patient T2 has been refractory to several courses of DNA-damaging treatments. In contrast, patient U1 received chlorambucil cures after these experiments were performed with a very good clinical response (Table I). T2 also presents poor prognosis-associated genetic alterations including a P53 mutation (Table I). More widespread genomic changes in both patients cannot be excluded. In B-CLL, single or complex abnormal genetic karyotypes develop with time. These abnormalities are rarely evident at clinical presentation and are clear examples of clonal evolution. The mechanisms that lead to the genomic alterations classically associated with B-CLL and to more widespread genomic changes are unknown. We suggest that error-prone DNA end joining may favour the emergence of genetic alterations and lesions in B-CLL. If a subset of B-CLL cells escape DNA damage- or genotoxic treatment-induced cell death and generate such genetic changes, then a wide array of functional variants might arise and could be selected to avoid clonal restraint and death. How does genome caretaker – classical NHEJ – become error prone in malignant B cells derived from a B-CLL patient refractory to treatment? This question of clinical relevance remains unresolved and deserves further investigation to establish whether genetic or post-translational modifications of one of the six known NHEJ components are involved. New protein associations may also contribute to the emergence of an error-prone NEHJ phenotype malignant B-CLL cells.
The authors are grateful to all the volunteer blood donors. We would like to thank Dr Hélène Salin (CEA-DRR-LRO, France) and Dr Florence Nguyen Khac (Hôpital Pitié-Salpétrière, France) for cytogenetic analysis, Dr Bernard Lopez (UMR CNRS/CEA 217, France) for helpful discussions and critical reading of the manuscript and Dr Johanne Bentley (St James's University Hospital, Leeds, UK) for pertinent comments.
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