Enhanced DNA-dependent protein kinase activity in Sjögren's syndrome B cells
G. Henriksson,
A. Sallmyr,
L. Du,
Å. Larsson1,
R. Manthorpe2 and
A. Bredberg
Department of Medical Microbiology, Lund University, Malmö University Hospital, 1 Department of Oral Pathology, Centre for Oral Health Sciences and 2 Department of Rheumatology, Sjögren's Syndrome Research Centre, Lund University, Malmö University Hospital, Malmö, Sweden.
Correspondence to: G. Henriksson, Department of Medical Microbiology, Malmö University Hospital, S-205 02 Malmö, Sweden. E-mail: gunnel.henriksson{at}mikrobiol.mas.lu.se
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Abstract
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Objective. To examine the stress response, including the role of DNA-dependent protein kinase (DNA-PK), in B cells from Sjögren's syndrome (SS) patients.
Methods. B-cell lines were exposed to gamma radiation and then postincubated to allow inducible stress functions to develop. The magnitude of the DNA damage response was monitored with respect to DNA-PK phosphorylation of a p53 peptide, defence protein levels (Ku, DNA-PK catalytic subunit, ATM, p21 and p53) and flow cytometric determination of cell cycle phases and apoptosis.
Results. B cells from SS patients, compared with healthy controls, displayed enhancement of two stress functions in undamaged cells: DNA-PK kinase activity and apoptosis. In addition, SS showed enhanced cell cycle arrest in gamma-irradiated cells.
Conclusions. Strong kinase activity of DNA-PK, functioning not only in a DNA damage response but also in immunoglobulin gene rearrangement, may be an important component of the heightened stress response displayed by SS cells. In combination with recent reports, our data indicate that constitutional hyper-reactivity to danger signals is a basic pathogenetic factor in SS.
KEY WORDS: Sjögren's syndrome, DNA-dependent protein kinase, Ku protein, Stress response, Cell cycle arrest
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Introduction
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Primary Sjögren's syndrome (SS) is characterized by diminished exocrine glandular function, autoantibody production and focal infiltration of B and T lymphocytes. Besides the cardinal symptoms of dry eyes and dry mouth, the disease may be complicated by systemic manifestations from the lungs, kidneys and nervous system, for example, as well as lymphoma [1, 2]. As for many other autoimmune diseases, the aetiology is unknown, but it is considered to be due to interaction between genetic and environmental factors [3]. The genetic factor is often thought to result in alterations in the immune system. However, a recent study showed that genes with a distinct expression pattern in autoimmunity are not necessarily immune response genes, but instead encode proteins involved in apoptosis, cell cycle progression, cell differentiation and cell migration [4].
Human cells can elicit a large set of defence functions for use in stress situations threatening cellular homeostasis, including DNA repair, cell cycle alterations and apoptosis [5, 6]. In previous studies of primary SS we have found alterations in such stress functions in SS lymphocytes; concerning DNA repair, DNA-binding proteins [79] and cell cycle arrest in T cells [10]. This has led to the hypothesis of an enhanced stress response as a pathogenetic mechanism in SS [11]. A major cellular component counteracting damage caused by gamma radiation and oxidative stress is DNA-dependent protein kinase (DNA-PK), composed of a catalytic subunit (DNA-PKcs) and the DNA end-binding heterodimeric Ku protein. Ku binds to double-stranded DNA breaks, leading to recruitment of DNA-PKcs to DNA, and activation of its kinase function. This initiates a cascade of events, including DNA-PK-mediated non-homologous end-joining of DNA strand breaks and, most importantly, kinase-dependent accumulation of p53 [12], modulating both cell cycle arrest in order to allow time for the cell to repair damage, and apoptosis eliminating severely damaged cells [13].
Interestingly, the DNA-PK complex, besides its role in DNA repair, forms an essential part of V(D)J recombinase, mediating the rearrangement of immunoglobulin and T cell receptor genes [14]. Accordingly, information on DNA-PK activity in B cells would be of special interest in an autoantibody-producing disease like SS. Therefore, in the present work we set out to analyse if an altered stress reaction also appears in B lymphocytes from SS patients, by determining DNA-PK activity, p53 phosphorylation, cell cycle progression and apoptosis. Our results show that strong DNA-PK kinase activity in B-cell lines is part of the enhanced stress reaction in SS cells, probably contributing to our observations of heightened cell cycle arrest and apoptosis.
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Materials and methods
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Patients and cells
Peripheral venous blood was obtained from four SS patients and two randomly selected healthy blood donors at the Malmö University Hospital blood bank. The patients (three positive and one negative for SS-A/SS-B autoantibodies) fulfilled both the USEuropean and the Copenhagen classification criteria for SS [15, 16]. B-cell lines were established from each individual by EpsteinBarr virus (EBV) transformation as described [7]. We also used one EBV-transformed B line (RA 143) derived from a healthy individual, obtained from Dr A. Auerbach, Rockefeller University, New York, USA. HeLa cells (2-CCL) were from the American Type Culture Collection (Rockville, MD, USA). All cells were cultured in RPMI 1640 medium (Gibco BRL, Paisley, UK) supplemented with 10% fetal calf serum, 2 mML-glutamine, 25 mM HEPES and 12 µg/ml gentamicin at 37°C in a humidified 5% CO2 atmosphere.
The design of the work conformed to the standards currently applied in Sweden at the time of the study and each subject gave her informed consent according to the Declaration of Helsinki before entering the study.
Ionizing irradiation
Gamma radiation was delivered by a neutron accelerator (Philips, Germany) at a dose rate of 0.70 Gy/min, at a distance of 50 cm from the cells, which were kept in their plastic culture flask in complete medium at room temperature. All cell cultures in the different assays were handled identically, the only difference being exposure to ionizing radiation. Following irradiation, cells were returned to the incubator and analysed later as indicated.
Extraction of nuclear proteins
Nuclear protein extracts were prepared by a modification of the procedure of Dignam et al. [17]. Cytoplasmic protein was removed by lysis of the cells in a low-salt buffer, and the sedimented cell nuclei were then lysed in a high-salt buffer, as described [10].
DNA-dependent protein kinase activity
The phosphorylating function of DNA-PK was measured using a synthetic p53 peptide (Promega, Madison, WI, USA) containing a DNA-PK specific phosphorylation site (serine 15) as substrate, as described previously [18]. Briefly, nuclear protein extract was mixed with [
-33P]ATP and the synthetic p53 peptide, with or without sonicated DNA. The DNA-PK activity was calculated as counts per minute (c.p.m.) in the presence of DNA minus the c.p.m. in the absence of DNA. HeLa cells were included in each analysis and used as a calibrator in order to reduce the interassay variation. All nuclear extracts were analysed in triplicate, with an intra-assay coefficient of variation (CV) of 8.7%. The interassay CV, estimated using HeLa cells and determinations made on seven separate dates, was 19.4%.
Western blotting
A standard immunoblotting was performed as described [18]. Membranes were probed with the following antibodies: anti-Ku86 (p80, Ab-3) (clone S10B1) and anti-p53 (Ab-2) (clone PAb1801) from Oncogene (Cambridge, MA, USA); anti-DNA-PKcs (C-19) (SC-1552) and anti-p21(F-5) (SC-6246) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-ATM (AHP397) from Serotec (Serotec, UK). The peroxidase-conjugated secondary antibodies were from Dako (DAKO A/S, Denmark). The blots were developed using the enhanced chemiluminescence method (ECL; Amersham-Pharmacia, UK), together with X-ray film or a Bio-Rad Personal Molecular Imager FX (Bio-Rad Laboratories, Hercules, CA, USA).
Cell cycle analysis
For cell cycle analysis a Coulter Epics XL-MCL flow cytometer (Coulter, Miami, FL, USA) was used. According to a standard total DNA labelling protocol the cells were incubated on ice for 1060 min in a hypotonic solution containing 0.01 M NaCl, 0.1% NP-40, 50 µg/ml propidium iodide and 10 µg/ml RNase A. To calculate the fractions of cells located in the different cell cycle phases MacCycle II software (Phoenix Flow, Phoenix, AZ, USA) was used and at least 8000 events were scored. For each individual three to six determinations were made at 24 h and one or two determinations 48 and 72 h after exposure to 15 Gy of irradiation.
Apoptosis
The same Coulter Epics XL-MCL flow cytometer was used, with filters detecting emission at 515545 nm for fluorescein isothiocyanate (FITC) and 565595 nm for propidium iodide. Background autofluorescence was monitored for each sample by analysing unlabelled cells. Apoptosis was measured as described [19], and at least 3000 events were scored. For each individual, two or three determinations were performed in unirradiated and 24 h post-irradiation cells. FITC-labelled Annexin V was from Alexis (Switzerland) and PharMingen.
Statistical analysis
The MannWhitney U-test was used for statistical analysis of the DNA-PK activity data, which included results from three controls and four SS patients. The lack of overlap between these two groups yielded a P value of less than 0.05 (0.028), considered statistically significant.
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Results
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High level of DNA-dependent protein kinase activity in SS
EBV-transformed B-cell lines were exposed to gamma radiation, post-incubated for 24 h under optimal growth conditions, and then subjected to nuclear protein extraction. DNA-PK kinase activity was determined by the incorporation of radiolabelled ATP into a synthetic p53 peptide containing a DNA-PK phosphorylation site. DNA-PK activity was calculated by subtracting the amount of radioactivity incorporated in the absence of sheared DNA from that incorporated in the presence of DNA. The assay takes advantage of the observation that DNA-PKcs appears to be the major DNA-activated kinase in mammalian cells [20]. The DNA-PK activity was higher in all four SS patients than in all three controls; i.e. there was no overlap between the groups (P = 0.028) (Fig. 1). The increased activity in SS was significant at all radiation doses, including unirradiated cells. There was no sign of induction of DNA-PK activity due to radiation exposure (except for the SS3 patient at 2 Gy); instead, a dose-dependent reduction was seen.

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FIG. 1. Enhanced DNA-PK-dependent activity in SS patients. The in vitro phosphorylation of a p53 synthetic peptide by B-cell nuclear protein extracts was analysed. Each circle represents one individual, and is the result of experiments performed in triplicate. C, control subjects; open circles, SS; filled circles, patients. The bar shows the median. The SS patient values are significantly higher at all three radiation doses, including unirradiated cells, than values for the controls (P = 0.028).
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Normal Ku86 and DNA-PKcs protein content in SS
The nuclear protein extracts were also used for Western blotting semiquantitation of Ku86 and DNA-PKcs protein content. SS and control samples, at each radiation dose, were electrophoresed on the same gel. The resulting bands suggest that there is no apparent difference between SS and controls, as shown by representative experiments in Fig. 2. These data do not permit evaluation of the influence of radiation on the protein level, since the extracts representing different radiation doses were analysed on separate immunoblotting gels. However, it has been reported that DNA-PK shows no inducibility in response to DNA damage [21].

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FIG. 2. Western blot determination of Ku86 and DNA-PKcs protein levels in gamma-irradiated and 24 h post-incubated B cells. Nuclear protein extracts were separated by SDSPAGE and immunoblotted. Results from representative experiments are shown. To facilitate quantitative comparison between SS and control samples, all samples from a given radiation dose were electrophoresed on the same gel.
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Enhanced cell cycle arrest due to gamma radiation in SS
At 24, 48 and 72 h after 15 Gy of irradiation, samples were drawn from the B-cell cultures and the DNA content was analysed by flow cytometry in order to determine cell cycle progression. Representative DNA histograms generated by the MacCycle software are shown in Fig. 3. The gamma radiation-induced response by the healthy controls is in agreement with that reported in the literature for EBV-transformed B-cell lines obtained from normal individuals [22, 23]. The reduction of cells located in G1 phase should be interpreted to result from two opposing influences: a G1 arrest function (under the influence of p53), together with a relatively strong loss of in-flow into G1 [24] resulting from DNA damage inhibiting DNA replication. This interpretation is supported by the findings of an intensified loss of G1 cells from ataxia telangiectasia (AT) patients known to be defective in ATM protein and cell cycle arrest function. Interestingly, cells from all the SS patients show a marked difference from the normal cells, contrasting with the abnormality reported in the AT cells, with a retention of G1 cells in response to gamma radiation (Fig. 4). This was observed at 24, 48 and 72 h after exposure to radiation. As expected, radiation can be seen to increase the fraction of sub-G1 events; i.e. cells or cell debris containing less than one DNA copy number. Some of these events can be assumed to represent cell death; see below for the apoptosis assay.

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FIG. 3. Cell cycle progression in gamma-irradiated B cells. DNA content was analysed by flow cytometry of propidium-labelled permeabilized cells. Representative experiments using cells incubated for 24 h after exposure to radiation are shown, with DNA histograms generated by the MacCycle software, referring the cells to cell cycle phases. The resulting value for the percentage of cells located in G1 is inserted into each histogram.
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FIG. 4. Increased G1 cell cycle arrest in SS patients. The fraction of cells located in the G1 phase, at the indicated times after exposure to radiation, was determined by flow cytometry, as shown in Fig. 3. Each symbol represents one individual, and shows the median value from three to six determinations at 24 h and one or two determinations at 48 and 72 h. Open circles, unirradiated controls; filled circles, unirradiated patients; open triangles, irradiated controls; filled triangles, irradiated patients.
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Normal p53, p21 and ATM protein levels in SS
p53 is known to activate transcription of p21, leading to inhibition of cell cycle progression from the G1 phase. Western blotting of nuclear protein extracts revealed induction of p53 and p21 upon gamma-irradiation in all cells examined; this is in accordance with the observation of G1 cell cycle arrest. ATM expression was unaffected by radiation, in agreement with the reported lack of inducibility of ATM [21]. There was no apparent difference between SS and healthy controls in the protein levels, as shown by a representative experiment in Fig. 5. However, it should be emphasized that this result does not exclude a difference in biological activity of these DNA damage response modifiers; compare our DNA-PK results in Figs 1 and 2 showing no effect on protein level but higher kinase activity in SS cells.

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FIG. 5. Western blot determination of p53, p21 and ATM protein levels in gamma-irradiated and 24 h post-incubated B cells. Nuclear protein extracts were separated by SDSPAGE and immunoblotted. Results from representative experiments are shown. Samples from all three radiation doses for each individual were electrophoresed on the same gel, facilitating evaluation of the influence exerted by radiation.
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Enhanced apoptosis in SS B cell lines
Early apoptosis was analysed by flow cytometry of the same radiation-exposed B-cell cultures as in the cell cycle experiments, and defined by the binding of annexin V to cells impermeable to propidium iodide. Results from representative experiments are visualized by the annexin V-positive but propidium iodide-negative events of the dot plot in the lower right quadrant (Fig. 6). The controls display a result similar to that reported in the literature for normal individuals, with a pre-irradiation value of approximately 10% apoptotic cells and becoming significantly increased by gamma radiation [23]. Two of the three SS lines differed markedly from this normal pattern even before irradiation, by having high median values for apoptotic cells (24 and 36% respectively) (Table 1). At 24 h after irradiation, the higher apoptosis frequencies were found in the SS cases (medians of 3471%) than in the controls (3335%). There was no apparent difference between SS and healthy controls in radiation-induced apoptosis; however, such a comparison is complicated by the very high apoptosis frequency (up to 71%) in irradiated SS cells (Table 1).

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FIG. 6. Apoptosis in gamma-irradiated (15 Gy) B cells, analysed by flow cytometry. Dot plots from representative experiments using unirradiated and 24 h post-incubated cells are shown, each dot representing one cell. Annexin V-positive, propidium iodide-negative cells (located in the lower right quadrant) are considered to be in apoptosis, whereas double-positive cells (upper right quadrant) are regarded as necrotic (or late apoptotic).
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Multiple facets of the stress response are enhanced in SS
An overview of the results of the present study is attempted in Table 1, by assigning a ranking number to each normal and SS individual, number 1 denoting the strongest defence reaction. The SS patients hold all the top positions in the pre-irradiation defence functions (DNA-PK kinase activity and apoptosis) as well as in the radiation-induced cell cycle arrest response (Table 1).
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Discussion
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Human cells express a number of complex and interacting mechanisms that constitute the stress response to DNA damage, including cell cycle arrest, allowing time for enzymatic DNA repair, and apoptosis of severely damaged cells. Some molecular components of these defence functions are expressed constitutively, whereas others become induced upon DNA damage formation. Double-stranded DNA breaks are thought to arise continuously in all cells, due to, for example, environmental radiation and endogenous radical oxygen species. Even a low frequency of this potent damage type, perhaps a single lesion per cell, is capable of inhibiting DNA replication and may threaten cellular homeostasis unless a stress response is evoked [5]. For the present work on the stress response in SS, we decided to focus on B cells and on gamma radiation-induced damage. Double-stranded DNA breaks occur physiologically in B cells during the rearrangement of immunoglobulin genes, and the DNA-PK complex participates in this recombination, not only in the bone marrow but also in mature B cells responding to antigen in lymph nodes [25]. Moreover, DNA-PK is essential for the repair of double-stranded DNA breaks by the mechanism of non-homologous end-joining, occurring mainly in G1, and also for the activation of the G1 arrest function mediated by p53 accumulation and p21 gene expression [5].
Accordingly, if a disease (such as SS) is found to have abnormalities in both immune regulation and stress response, we reasoned that the molecular basis may be shared between these two processes, DNA-PK being a likely candidate. EBV-transformed, continuously growing B-cell lines were used, since sufficient peripheral blood B cells could not be obtained from patients. In a previous study, Zeher et al. [26] found that the increased susceptibility to apoptosis of peripheral blood CD4+ T cells from SS patients correlates with lymphocyte activation. We find it unlikely that our EBV lines, cultivated for several weeks and for more than ten generations in vitro at the time of these experiments, should retain an imprint of in vivo activation status. Therefore, we considered it wise to avoid primary B cells, acknowledging that SS-related in vivo activation of B cells might influence any result in a stress response study.
To what extent is the cellular stress response affected by the status of EBV transformation and its accompanying growth alterations? Infection of human B lymphocytes with EBV in vitro induces a G0 to G1 transition followed by DNA synthesis and cell division. This cell cycle activation closely mimics the antigen-dependent normal B-cell activation pathway and is mediated by the virus-encoded latent membrane protein 1 (LMP1), which is a functional homologue of CD40, and LMP2, which mimics the survival signal that is usually provided by the B-cell antigen receptor [27]. Significant interaction between EBV and a stress response factor such as p53 or DNA-PK, to the best of our knowledge, has not been demonstrated. Furthermore, G1 arrest function with p53 stabilization and p21 production has been reported in gamma-irradiated EBV lines [23]. Therefore, we consider B-cell lines to be an approximation of normal antigen-stimulated B cells, with no difference to be anticipated between SS patients and controls in activation level. Even though the number of cell lines studied is small, we regard the results as representative of SS since the patients fulfil appropriate criteria for inclusion, and the observed differences between patients and controls were distinct, with no overlapping.
The present study demonstrates an enhanced stress response capacity in SS B-cell lines. Two of the defence functions we assayed were found to be enhanced in undamaged cells: (i) the capacity of DNA-PKcs to become activated in vitro by broken DNA and phosphorylate a p53 peptide; and (ii) apoptosis. The third stress function assayed, G1 cell cycle arrest, depends on the accumulation of p53 being induced by DNA damage. Therefore, our observation of an elevated G1 arrest function illustrates an enhanced capacity in SS cells also in a stress-induced defence function. The evaluation of cell cycle data in proliferating cell lines is complicated by the induction of DNA damage, and possibly also arrest mechanisms, in all cycle phases. For example, a deficient S/G2 arrest may simulate enhanced G1 arrest by the exportation of cells into the next-coming G1, and thereby increase the percentage of G1 cells. However, our interpretation of enhanced G1 arrest in the SS lines is supported by reports of AT cells lacking the ATM stress regulatory protein, leading to deficient cell cycle arrest in response to gamma radiation [28]. EBV lines from AT patients display features which are strikingly opposite to those found by us in SS [2224]. Thus, radiation causes the percentage of AT cells in G1 to decrease more than in controls, and apoptosis is reduced. Also, in AT blood lymphocytes the frequency of chromosome translocations mediated by V(D)J recombinase is increased [29], whereas we have reported a lack of translocations in SS patients [9]. This comparison with AT supports the notion that the present SS findings reflect hyperactive G1 arrest.
A number of reports on both salivary glands and blood lymphocytes, when combined, may be taken to suggest a constitutional predisposition in SS to over-react when confronted with stress stimuli. These reports include DNA repair alterations [30, 31], apoptosis dysregulation [32, 33], elevated cellular DNA damage response [8], up-regulation of cell cycle inhibition [10], and protein kinase C signalling abnormalities [34]. A rationale for the salivary gland to be a locus minoris resistentiae to stress signals has recently been outlined by Konttinen et al. [35], based on a unique tissue architecture and on continuous exposure to ascending microorganisms and other foreign materials. In support of this viewthe salivary gland being a vulnerable organexperimental evidence of a constitutively high level of stress has been provided by demonstration of DNA strand breaks and DNA repair Ku protein in ductal cells [36], as well as by extremely high radiosensitivity of salivary glands (C. Barlow, personal communication). Consequently, in the salivary gland reactivity to stress and DNA damage seems to be of great significance. Interestingly, our observations of a tendency in SS patients to display a heightened genotoxic response are in agreement with previous reports on the overexpression of wild-type p53 and its transcription factor p21 in labial salivary glands [37, 38].
In summary, our present results suggest that DNA-PK activity is part of a heightened stress response in SS B cells involving cell cycle arrest and apoptosis. Because of a role of DNA-PK also for immunoglobulin rearrangement, our findings raise the question of whether the DNA-PK alteration influences B-cell maturation and, thus, the development of autoimmunity features. B-cell activating factor, secreted by macrophages and dendritic cells, has recently become strongly linked to the development of SS in both man and mice [39]. It is therefore intriguing to consider whether an elevated stress response is a primary aetiological factor in SS [11], leading to hyper-alert innate immunity, and also to derangement of adaptive immunity, as predicted by the Danger model for lymphocyte activation [40].
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Acknowledgments
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This work was supported by grants from the Swedish Institute, the Swedish Cancer Society, Malmö University Hospital Funds, and the Foundations of Osterlund, Gustaf V:s 80arsfond, Greta och Johan Kock, Crafoord and Gunnar Nilsson. We are grateful to Anders Nilsson and Asa Eriksson for advice on DNA-PK activity measurement, and to Jan Ake Nilsson for expert statistical assistance.
The authors have declared no conflicts of interest.
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References
|
---|
- Jonsson R, Haga H-J, Gordon TP. Sjögren's syndrome. In: Koopman WJ, ed. Arthritis and allied conditions: A textbook of rheumatology, 14 edn. Baltimore: Lippincott, Williams & Wilkins, 2000:182649.
- Kassan S, Thomas T, Moutsopoulos H et al. Increased risk of lymphoma in sicca syndrome. Ann Intern Med 1978;89:88892.[ISI][Medline]
- Price EJ, Venables PJW. The etiopathogenesis of Sjögren's syndrome. Semin Arthritis Rheum 1995;25:11733.
- Maas K, Chan S, Parker J et al. Cutting edge: molecular portrait of human autoimmune disease. J Immunol 2002;169:59.[Abstract/Free Full Text]
- Jackson SP. Sensing and repairing DNA double-strand breaks. Carcinogenesis 2002;23:68796.[Abstract/Free Full Text]
- Sionov RV, Haupt Y. The cellular response to p53: the decision between life and death. Oncogene 1999;18:614557.[CrossRef][ISI][Medline]
- Henriksson G, Sandor Z, Aponyi I, Manthorpe R, Bredberg A. A reduced level of multiple mutation in a shuttle vector passaged in Sjögren's syndrome cells. Mutat Res 1994;324:10310.[CrossRef][ISI][Medline]
- Lavasani S, Henriksson G, Brant M et al. Abnormal DNA damage-inducible protein in cells from Sjögren's syndrome patients. J Autoimmun 1998;11:3639.[CrossRef][ISI][Medline]
- Henriksson G, Brant M, Sandor Z, Manthorpe R, Bredberg A. Sjögren's syndrome: lymphoma predisposition coupled with a reduced frequency of t(14;18) translocations in blood lymphocytes. Mol Carcinog 1999;24:22631.[CrossRef][ISI][Medline]
- Henriksson G, Brant M, Sallmyr A, Fukushima S, Manthorpe R, Bredberg A. Enhanced DNA damage-induced p53 peptide phosphorylation and cell-cycle arrest in Sjögren's syndrome cells. Eur J Clin Invest 2002;32:45865.[CrossRef][ISI][Medline]
- Bredberg A, Henriksson G, Larsson A, Sallmyr A, Manthorpe R. A role of the macrophage in Sjögren's syndrome? Scand J Rheumatol 2003;32:255.[CrossRef][ISI][Medline]
- Smith GCM, Jackson SP. The DNA-dependent protein kinase. Genes Dev 1999;13:91634.[Free Full Text]
- Gudkov AV, Komarova EA. The role of p53 in determining sensitivity to radiotherapy. Nature Rev 2003;3:11729.[CrossRef][ISI]
- Gellert M. Recent advances in understanding V(D)J recombination. Adv Immunol 1997;64:3964.[ISI][Medline]
- Vitali C, Bombardieri S, Jonsson R et al. Classification criteria for Sjögren's syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis 2002;61:5548.[Abstract/Free Full Text]
- Manthorpe R, Oxholm P, Prause JU, Schiødt M. The Copenhagen criteria for Sjögren's syndrome. Scand J Rheumatol 1986;61(Suppl.):1921.
- Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 1983;11:147589.[Abstract]
- Sallmyr A, Henriksson G, Fukushima S, Bredberg A. Ku protein in human T and B lymphocytes: full length functional form and signs of degradation. Biochim Biophys Acta 2001;1538:30512.[CrossRef][ISI][Medline]
- Sallmyr A, Du L, Bredberg A. An inducible Ku86-degrading serine protease in human cells. Biochim Biophys Acta 2002;1593:5768.[CrossRef][ISI][Medline]
- Finnie NJ, Gottlieb TM, Blunt T, Jeggo PA, Jackson SP. DNA-dependent protein kinase activity is absent in xrs-6 cells: implications for site-specific recombination and DNA double-strand break repair. Proc Natl Acad Sci USA 1995;92:32024.[Abstract]
- Yang J, Yu Y, Duerksen-Hughes PJ. Protein kinases and their involvement in the cellular responses to genotoxic stress. Mutat Res 2003;543:3158.[ISI][Medline]
- Naeim A, Repinski C, Huo Y et al. Ataxiatelangiectasia: flow cytometric cell-cycle analysis of lymphoblastoid cell lines in G2/M before and after gamma-irradiation. Modern Pathol 1994;7:58792.[ISI][Medline]
- Meijer AE, Zhivotovsky B, Lewensohn R. EpsteinBarr virus-transformed lymphoblastoid cell lines of ataxia telangiectasia patients are defective in X-ray-induced apoptosis. Int J Radiat Biol 1999;75:70916.[CrossRef][ISI][Medline]
- Skog S, Lewensohn R, He Q, Borg AL, Gatti R. Kinetics of G1/S and G2/M transition in x-irradiated ataxiatelangiectasia cells. Cancer Detect Prev 1997;21:91102.[ISI][Medline]
- Han S, Dillon SR, Zheng B, Shimoda M, Schlissel MS, Kelsoe G. V(D)J recombinase activity in a subset of germinal center B lymphocytes. Science 1997;278:3014.[Abstract/Free Full Text]
- Zeher M, Szodoray P, Gyimesi E, Szondy Z. Correlation of increased susceptibility to apoptosis of CD4+ T cells with lymphocyte activation and activity of disease in patients with primary Sjögren's syndrome. Arthritis Rheum 1999;42:167381.[CrossRef][ISI][Medline]
- Thorley-Lawson DA. EpsteinBarr virus: exploiting the immune system. Nature Rev Immunol 2001;1:7582.[CrossRef][Medline]
- Shiloh Y. ATM and ATR: networking cellular responses to DNA damage. Curr Opin Genet Dev 2001;11:717.
- Lipkowitz S, Stern MH, Kirsch IR. Hybrid T cell receptor genes formed by interlocus recombination in normal and ataxia-telangiectasia lymphocytes. J Exp Med 1990;172:40918.[Abstract]
- Guo K, Major G, Foster H et al. Defective repair of O6-methylguanine-DNA in primary Sjögren's syndrome patients predisposed to lymphoma. Ann Rheum Dis 1995;54:22932.[Abstract]
- Bashir S, Harris G, Denman MA, Blake DR, Winyard PG. Oxidative DNA damage and cellular sensitivity to oxidative stress in human autoimmune diseases. Ann Rheum Dis 1993;52:65966.[Abstract]
- Humphreys-Beher MG, Peck AB, Dang H, Talal N. The role of apoptosis in the initiation of the autoimmune response in Sjögren's syndrome. Clin Exp Immunol 1999;116:3837.[CrossRef][ISI][Medline]
- Bolstad AI, Eiken HG, Rosenlund B, Alarcon-Riquelme ME, Jonsson R. Increased salivary gland tissue expression of Fas, Fas ligand, cytotoxic T lymphocyte-associated antigen 4, and programmed cell death 1 in primary Sjögren's syndrome. Arthritis Rheum 2003;48:17485.[CrossRef][ISI][Medline]
- Törnwall J, Konttinen YT, Tuominen RK, Törnwall M. Protein kinase C expression in salivary gland acinar epithelial cells in Sjögren's syndrome. Lancet 1997;349:18145.[ISI][Medline]
- Konttinen YT, Käsnä-Ronkainen L. Sjögren's syndrome: viewpoint on pathogenesis. Scand J Rheumatol 2002;Suppl. 116: 1522.[CrossRef]
- Larsson A, Henriksson G, Manthorpe R, Sallmyr A, Bredberg A. Ku protein and DNA strand breaks in lip glands of normal and primary Sjögren's syndrome subjects: lack of correlation with apoptosis. Scand J Immunol 2001;54:32834.[CrossRef][ISI][Medline]
- Mariette X, Sibilia J, Roux S, Meignin V, Janin A. A new defensive mechanism to prevent apoptosis in salivary ductal cells from patients with Sjögren's syndrome: over-expression of p53 and p21. Rheumatology 2002;41:969.[Abstract/Free Full Text]
- Tapinos NI, Polihronis M, Moutsopoulos HM. Lymphoma development in Sjögren's syndrome. Novel p53 mutations. Arthritis Rheum 1999;42:146672.[CrossRef][ISI][Medline]
- Groom J, Kalled SL, Cutler AH et al. Association of BAFF/BLyS overexpression and altered B cell differentiation with Sjögren's syndrome. J Clin Invest 2002;109:5968.[Abstract/Free Full Text]
- Matzinger P. The danger model: a renewed sense of self. Science 2002;296:3015.[Abstract/Free Full Text]
Submitted 13 January 2004;
revised version accepted 13 May 2004.