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Carcinogenesis |
Department of Haematological Medicine, Leukaemia Sciences Laboratories, The Rayne Institute, Guy’s, King’s, St. Thomas’ School of Medicine, Denmark Hill, London SE5 9NU, United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20 bp) back to regions of microhomology of 1–6 bases (11
, 12)
. Despite the potential role of the NHEJ system in genomic instability in cancer, little is understood of the mechanism by which it participates in cancer. In fact, previous reports have been confusing in this regard because both deficiencies and increased NHEJ activity have been reported to mediate genomic instability. For example, recent studies of mouse cells null for components of the NHEJ pathway suggest the importance of these proteins for protecting against genetic instability and tumorigenesis (reviewed Ref. 13 ). Ku86-/- mice have a marked increase in chromosomal instability manifesting as breakage, translocation, and aneuploidy and a significant incidence of B-cell lymphoma (14) . On the other hand, several lines of evidence suggest that cells with an intact NHEJ pathway can give rise to chromosomal translocations and deletions in the repair of DSBs (15 , 16 ; reviewed in Refs. 4 , 17 ). Recently, Rothkamm et al. (15) showed that if multiple DSBs are generated in cells wild type for NHEJ proteins, a high frequency of misjoining and genomic rearrangements are detected. In fact, these authors showed a dramatic decrease in genomic rearrangements and misrepair in cell lines defective for NHEJ (15 , 16) . We previously demonstrated that nuclear extracts prepared from the Bloom’s chromosomal instability cells and myeloid leukemias show a significant increase in end-ligation efficiency and frequency of misrepair, as compared with normal hematopoietic cells (18 , 19) . Thus, the above, seemingly contradictory, findings associated with chromosome instability suggest that the role of NHEJ in cancer is complex and that underactivity as well as overactivity could contribute to DNA repair infidelity in neoplastic cells.
We present additional evidence for the role of increased NHEJ activity in creating genomic instability during the repair of DSBs. We show how these increases may result from cellular responses to both induced DSBs in normal cells and constitutive damage in myeloid leukemia cells, thus presenting a mechanism that may account for the increased aberrant NHEJ activity in these cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mobilized peripheral blood and bone marrow progenitor cells (CD34+) were harvested from normal and healthy donors and were cultured as detailed previously (18 , 19) . PBLCs from normal subjects were prepared as described previously (18 , 19) . Newly diagnosed and untreated myeloid leukemia patient samples were received from hematology clinics. Clinical diagnosis and cytogenetics analysis were made on each sample before primary cell harvesting using Hypaque-Ficoll gradients. The mononuclear fraction was isolated from 10–20 ml of chronic phase CML or AML peripheral blood. Cytospins of these fractions were examined morphologically after May-Grunwald Giemsa staining and revealed the presence of >95% of CML cells or AML blasts, respectively; lymphocyte and monocyte contamination were negligible. Primary cells were cultured at 1 x 106/ml in complete medium for 24 h before nuclear extraction. Typically, between 2 x 106–107 cells were used for the preparation of nuclear extracts.
Cells were treated agents at concentrations used previously to elicit chromosome breakage (20)
. Log-phase cells were irradiated on ice in a Gammacell Cobolt 60 
irradiator at doses between 3–50 (6.52 Gy/min). Cells were then returned to culture in fresh prewarmed media for a period of 30 min. Log-phase cells were cultured in 0.5 µg/ml aphidicolin (Sigma) and left in culture for 8–14 h. Thereafter, nuclear extracts were prepared. Log-phase cells treated with busulphan (5 µg/ml) were treated in a similar fashion.
Preparation of nuclear extracts and procedures for the end ligation and plasmid reactivation assays have been described previously (18 , 19) .
Plasmids and Antibodies.
pUC18 was linearized with EcoRI (MBI Fermentes, Cleveland, United Kingdom), dephosphorylated with calf intestine alkaline phosphatase (Promega, Southampton, United Kingdom), and 32P-labeled with T4 polynucleotide kinase (Promega). Goat polyclonal antisera raised against Ku86, Ku70, and DNA-PKcs and Oct-2 and their respective blocking peptides were purchased from Santa Cruz Technologies (Santa Cruz, CA). Ku86 and Ku70 blocking peptides mapped to the COOH terminus (amino acids 713–730 and 590–608) of their respective proteins. Rabbit polyclonal antisera raised against Rad51 was a kind gift from Stephen West (Imperial Cancer Research Fund, South Mimms, United Kingdom). Mouse monoclonal antibodies to BrdUrd (Becton Dickinson) were used for DNA damage assays according to manufacturer’s protocols.
It has been previously reported that cell extracts possess nuclease activity that degrades the input plasmid DNA having an antagonistic effect on end-joining efficiency (21) . The preparation of nuclear extracts alleviated this problem. We confirmed that the end-joining efficiencies observed in our assays were not the result of varying nuclease activity, by incubating nuclear extracts from different sources with linearized and labeled plasmid DNA without end ligation buffer for 24 h at 18°C. All experiments were performed in the linear phase of the end-ligation reaction, and each sample was tested for significant nuclease activity that could influence end-ligation results; no significant nuclease activity was present in our samples.
Antibody Abrogation Studies.
For antibody abrogation studies, dilutions of antisera (100 µg/ml) were incubated in the reaction mixture for 5 min at 37°C before incubation for 24 h at 18°C. Antibodies were blocked with 5-fold excess (by weight) of blocking peptide or blocking molecule (see below, anti-BrdUrd block with BrdUrd) in a small volume of PBS. The blocking reaction was incubated overnight at 4°C.
DNA Damage Studies.
These studies were performed according to the protocols of Raderschall et al. (22)
. Cells were grown in BrdUrd (10 µM) for 
30 h and were shielded from light. Thereafter, the cells were washed and placed in BrdUrd-free medium for 1 h. To induce DNA damage, cells were exposed to ionizing irradiation, aphidicolin, or busulphan (see above) before washing in BrdUrd-free medium for 1 h. Cells were then washed twice in PBS and cytospun onto glass slides, and preparations were fixed in absolute methanol for 30 min at -20°C and then rinsed in ice-cold acetone for up to 1 min. Chromatin fibers were prepared from 1 x 106 cells, according to protocols of Raderschall et al. (22)
. However, we modified the protocol to ensure subsequent detection of proteins binding at specific sites of interest. Proteins were cross-linked to DNA by adding formaldehyde (1% final concentration) to the culture medium for 10 min at 37°C. Aliquots of 1 x 106 cells were trypsinized and the cells cytospun onto glass slides and covered with 50 µl of 50 mM Tris HCl (pH 8), 1 mM EDTA, and 0.1% SDS. After 1-min incubation with the detergent solution, the chromatin was mechanically sheared on the slide with the aid of a glass coverslip and then fixed with methanol and acetone (as described above).
Immunofluorescence Detection.
For detection of ssDNA inside the nucleus, cytospin preparations of BrdUrd-substituted cells were processed without a prior denaturation step. Slides were incubated with blocking solution (10% BSA/4 x SSC/0.1% Tween 20) for 30 min at 37°C. Thereafter, slides were incubated with primary anti-BrdUrd (Becton Dickinson) diluted in blocking serum (1/10–1/50) and incubated for 30 min at 37°C. Slides were washed for 5 min in 4 x SSC/0.1% Tween 20 and repeated two more times. The blocking step was then repeated before slides were incubated with secondary antibody conjugated with fluorochromes diluted in blocking solution (1/200; Sigma) and subsequently washed as above. Cells were counterstained with DAPI for 1 min, rinsed in PBS, and coverslips were mounted in antifade solution ready for analysis. Control experiments were performed using anti-BrdUrd prebound to BrdUrd. In addition, cells not incorporated with BrdUrd were also used as controls. The slides were examined using Olympus fluorescent microscope with DAPI/FITC/rhodamine triple pass filters, and images were captured using a charge-coupled device camera and software (Smart Capture VP; Digital Scientific Ltd., Cambridge, United Kingdom) and data analyzed (Quips XL, Vysis, Inc., Surrey, United Kingdom).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Table 1
). However, despite the small effects of treatment, the percentage of aberrant large plasmid deletions examined from these misrepair experiments was significantly increased in exposed normal cells, compared with untreated counterparts [33 versus 4%, mean (n = 6), P < 0.001; Fig. 1 E–G
]. These data suggest that in response to excessive DSBs, normal hemopoietic cells have a similar pattern but lower level of NHEJ activity and misrepair, compared with untreated myeloid leukemia cells.
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myeloblastic), and K562 (CML erythroleukemia); Refs. 23
, 24
] produced small but reproducible overall increases in mean end-ligation activities (Fig. 2, A–F)
. Primary samples [five AMLs (3 FAB M4, 1 M2, 1 M5) and three CMLs at presentation] also produced small but consistent increases in end-ligation efficiencies [AML, 51–63%; CML, 46–57%; mean (n = 3)]. The misrepair frequencies in myeloid leukemia cell lines after DSB induction showed small increases, with ionizing radiation generating up to 2-fold increases in HL60 cells (Fig. 3, A and B)
. Misrepair frequencies for primary samples were consistent with data for the cell lines [AML, 7–15%; CML, 5.8–11%, mean (n = 3); Table 1
]. Analysis of the size of plasmid deletions by PCR clearly showed a higher frequency of aberrant deletions compared with untreated cells, corroborating the evidence for increased misrepair in these cells [90% treated versus 85% untreated, mean (n = 6); Fig. 3, C–E
].
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and 3, A and B
; Table 1
). This result was additionally confirmed by PCR analysis of misrepaired colonies that showed a lower frequency of large deletions than demonstrated for radiation and aphidicolin-treated cells (Fig. 3D)
.
Increased End-Ligation Efficiency and Misrepair in both Myeloid Leukemias and Normal Cells in Response to DSBs Is Dependent on NHEJ Proteins.
To confirm that proteins involved in NHEJ are responsible for the increased end-ligation efficiencies and DSB misrepair observed after DSB induction, nuclear extracts from the normal (PBLC) and myeloid leukemia cells exposed to irradiation (6 Gy) were preincubated with antibodies to Ku70 and Ku86 (diluted 1/200–1/10). Thereafter, both end-ligation efficiency and misrepair assays were performed as above. We find that there is a decrease in the end-ligation efficiency (Fig. 4A)
and DSB misrepair frequency (Fig. 4B)
with increasing concentration of Ku70 and Ku86 antibodies, with both myeloid leukemia and normal cells. Control antibodies for proteins such as Rad51 and antibodies to Ku86 and Ku70 prebound to their cognate peptides showed no decrease in misrepair frequency with increasing antibody concentration. These results show that DSB end-ligation activity and misrepair in both normal and myeloid leukemia cells after DSB induction is dependent on the presence of Ku70 and Ku86 in nuclear extracts.
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. Analysis of DNA sequences at the breakpoint junction demonstrated regions of microhomology in all eight clones (Table 2)
. This is additional evidence for the participation of NHEJ in the repair of these plasmid DSB in vitro.
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We directly tested the levels of DNA damage in normal and myeloid leukemia cells using an assay that has previously been used to detect DNA damage after treatment with DSB-inducing agents, and the Rad 51 DSB repair protein has previously been localized to these sites (22)
. This assay relies on the specificity of anti-BrdUrd for ssDNA after treatment of BrdUrd-incorporated cells with DNA damaging agents. It is thought that after formation of a DSB, this assay detects the ssDNA created to expose DNA to repair proteins and thus can be considered a marker for this DNA damage. Thus, ssDNA regions at the sites of DSB can be visualized and quantified as very bright BrdUrd-positive nuclear foci. We, thus assessed DNA damage in normal myeloid leukemic and normal cells after treatment with radiation, aphidicolin, and etoposide. To confirm previous data, we exposed normal hematopoietic cells [IL-2-stimulated T lymphocytes (n = 4), CD34+ cells (n = 3)] to the DSB-inducing agents (ionizing radiation, aphidicolin, and etoposide). Treatment with these agents results in a significant increase in cells containing DNA damage foci [7–26%, mean (n = 3), P < 0.001; Table 2
]. In contrast, control experiments where anti-BrdUrd was preincubated with BrdUrd showed no damage foci (Fig. 5)
. Strikingly, even in the absence of treatment with DNA damaging agents, myeloid leukemia cell lines (HL60 and K562) and primary cells [CML (n = 2)] show low but significant levels of constitutive DNA damage compared with normal hemopoietic cells [IL-2-stimulated T lymphocytes (n = 4), CD34+ cells (n = 3); 34 versus 7%, mean (n = 3), P < 0.001; Table 3
, Fig. 5
]. In contrast, myeloid leukemia cells treated with busulphan demonstrated a pattern of DNA damage foci similar to untreated cells (Table 2
, Fig. 5
). Normal cells treated with busulphan also appear to have less DNA damage compared with counterparts treated with DSB-inducing agents (Table 3
, Fig. 5
).
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. Chromatin fibers were prepared from BrdUrd-incorporated normal (PBLC) and myeloid leukemic cells (HL60 and K562) before and after treatment with ionizing radiation (6 Gy). Between 15 and 20 fibers were examined for BrdUrd-positive regions of DNA damage in each cell preparation. These experiments were performed three times. Although fibers from untreated normal cells showed no BrdUrd-positive regions, untreated myeloid leukemia cells demonstrated fibers containing DNA damage (K562, 20%; HL60, 26%). After ionizing radiation, chromatin fibers from normal cells demonstrate DNA damage (12%), and fibers from myeloid leukemias, in general, appear to contain more damage, compared with untreated counterparts. However, the variability in the stretch of fibers precluded definitive quantitation of the perceived increased damage in treated myeloid leukemia cells (Fig. 6A)
.
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]. These data provide evidence that both the constitutively expressed damage in myeloid leukemia cells and induced damage in normal cells are DSBs, colocalizing with proteins intrinsic to the NHEJ pathway. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A striking finding of this study is that normal cells exposed to agents that induce DSBs demonstrate a similar pattern of NHEJ activity and misrepair to frank leukemia cells, albeit at much lower levels. Irradiation and, to a lesser extent, aphidicolin treatment generate increased frequencies of aberrant NHEJ, most likely in accordance with their ability to induce DSBs (25)
. In contrast, alkylating agents, which generate a minimal amount of DSBs, demonstrate little or no effect on NHEJ activity and misrepair (25)
. These findings suggests that excessive DNA damage may force normal NHEJ components to process DSB aberrantly, resulting in possible chromosomal instability.
An important ramification of our findings relates to causative effects of human leukemia and how the disease may progress. Our data suggest that genomic instability may actually start in normal cells challenged to respond to damage by DSB-inducing agents. Several lines of evidence suggest that a major consequence to health of exposure to ionizing radiation is genomic instability and leukemia (26) . For example, leukemias that arose in atomic bomb survivors and radiation therapy-related leukemias exhibit high levels of chromosome abnormalities (27 , 28) . This data is in keeping with the idea that the type of genomic instability generated in radiation-induced leukemias is associated with the repair of DSBs, of which NHEJ is a major participant. Therefore, the increased NHEJ activity and consequent misrepair we have observed is an attractive candidate for the generation of chromosomal instability in myeloid leukemias in vivo.
A key aspect of our present work is to use a whole cellular context to tie constituents of the NHEJ repair complex to the increased damage misrepair in leukemia cells. In addition to titration studies with antibodies to key NHEJ repair proteins that strongly indicate in our in vitro assays that infidelity of accompanying DSB repair is dependent on the presence of Ku70 and Ku86, we directly visualize these proteins at ongoing sites of damage in leukemia cells and at such sites arising in response to DSB-inducing agents. Although the basis for the repair infidelity of the Ku70/86 heterodimer is unclear, our data suggest that the normal NHEJ apparatus is capable of DSB misrepair when challenged with excessive DSBs. Our direct sequencing analyses of plasmid deletions generated in normal cells after DSB induction suggests that the repair occurred through ligation of distant regions of microhomology, a characteristic of the NHEJ mechanism. Furthermore, our data are in keeping with recent studies in normal eukaryotic cells that demonstrate NHEJ can generate genomic rearrangements when DSBs are induced (15 , 16) .
Much remains to be learned about the mechanisms through which increased misrepair NHEJ activity arises in myeloid leukemias. Examination of proteins specifically involved in this poorly understood misrepair pathway will additionally elucidate its role and activation in response to DNA damage. Furthermore, the structure and phosphorylation of Ku and Ku binding proteins will provide additional insights into misrepair activity in myeloid leukemias. However, the high frequency of chromosome abnormalities in cancer cells is likely to result, in part, from a high rate of error-prone NHEJ at regions of DSBs, which may be generated in response to increased levels of endogenous DNA damage in these cells. Our data indicate that this is a process that could be fundamental to the risk of developing diseases like leukemia and to all stages of progression of such malignancies once they become established.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Elimination of Leukaemia Fund (to F. V. R., T. J. G., N. B., M. C.). 
2 These authors contributed equally to the work.
3 To whom requests for reprints should be addressed, at Department of Haematological Medicine, Leukaemia Sciences Laboratories, The Rayne Institute, GKT, Denmark Hill, London SE5 9NU, United Kingdom. Phone: 44-207-848-5821; Fax: 44-207-848-5814; E-mail: feyruz.rassool{at}kcl.ac.uk
4 The abbreviations used are: DSB, double strand break; DNA-PKcs, DNA-protein kinase; PBLC, peripheral blood lymphocyte; CML, chronic myelogenous leukemia; AML, acute myelogenous leukemia; BrdUrd, bromodeoxyuridine; ssDNA, single-stranded DNA; DAPI, 4',6-diamidino-2-phenylindole; IL, interleukin.
Received 10/10/02. Accepted 2/19/03.
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gene, and the purified DNA was used to transform Escherichia coli. The frequency of misrepair was calculated by counting the number of white (misrepaired) colonies as a percentage of the total (blue + white) colonies. E, mean percentage of aberrant deletions detected in PCR products from misrepaired colonies after PBLC irradiation or aphidicolin treatment. Twenty to 50 misrepaired (white) colonies were analyzed/experiment (n = 4). F and G, PCR products from PBLCs, untreated (W1–W4), irradiated (w5–10), aphidicolin-treated (W11–W17). Blue colony (b), white colony (W), L = DNA ladder (bp).
