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The Journal of Neuroscience, August 29, 2007, 27(35):9451-9457; doi:10.1523/JNEUROSCI.0523-07.2007
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Cellular/Molecular
Repair Capacity for Platinum-DNA Adducts Determines the Severity of Cisplatin-Induced Peripheral Neuropathy
Anna Dzagnidze,1 Zaza Katsarava,2 Julia Makhalova,1 Bernd Liedert,1 Min-Suk Yoon,2 Holger Kaube,3,4 Volker Limmroth,2 andJuergen Thomale1 1Institut für Zellbiologie (Tumorforschung) and 2Klinik für Neurologie, Universitätsklinikum der Universität Duisburg-Essen, D-45122 Essen, Germany, 3Department of Neurology and Neurophysiology, University of Freiburg, D-79095 Freiburg, Germany, and 4Division of Neurosciences, Medical School, Southampton University, Southampton SO16 64D, United Kingdom
Abstract
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Abstract
Introduction
The pronounced neurotoxicity of the potent antitumor drug cisplatin frequently results in the onset of peripheral polyneuropathy (PNP), which is assumed to be initially triggered by platination products in the nuclear DNA of affected tissues. To further elucidate the molecular mechanisms, we analyzed in a mouse model the formation and processing of the main cisplatin-induced DNA adduct (guanine–guanine intrastrand cross-link) in distinct neuronal cell types by adduct-specific monoclonal antibodies. Comparison of the adduct kinetics in cisplatin-injected mice either proficient or deficient for nucleotide excision repair (NER) functions revealed the essential role of this DNA repair pathway in protecting differentiated cells of the nervous system from excessive formation of such lesions. Hence, chronic exposure to cisplatin resulted in an accelerated accumulation of unrepaired intrastrand cross-links in neuronal cells of mice with dysfunctional NER. The augmented adduct levels in dorsal root ganglion (DRG) cells of those animals coincided with an earlier onset of PNP-like functional disturbance of their sensory nervous system. Independently from the respective repair phenotype, the amount of persisting DNA cross-links in DRG neurons at a given cumulative dose was significantly correlated to the degree of sensory impairment as measured by electroneurography. Collectively, these findings suggest a new model for the processing of cisplatin adducts in primary neuronal cells and accentuate the crucial role of effectual DNA repair capacity in the target cells for the individual risk of therapy-induced PNP.
Key words: DNA adducts; repair; cisplatin; polyneuropathy; monoclonal antibodies; dorsal root ganglion
Introduction
Cisplatin (cis-diamminedichloroplatinum) is a widely used and effective anticancer drug. Treatment, however, is associated with several side effects such as nephrotoxicity and neurotoxicity (Mollman, 1990). Although damage of kidney tubular epithelium cells can be avoided by forced diuresis, peripheral neurotoxicity remains a severe clinical problem and a major dose-limiting factor of cisplatin therapy (LoMonaco et al., 1992
The antineoplastic activity of cisplatin is based on the formation of platination products in the nuclear DNA (Boulikas and Vougiouka, 2003
The precise mechanisms of cisplatin-induced peripheral neurotoxicity and the onset of PNP are still obscure. Although there is increasing evidence that the formation of DNA platination products in dorsal root ganglion (DRG) cells is the main trigger for the malfunction of the sensory nervous system (Meijer et al., 1999
The aim of the current study was to test the hypothesis whether the levels of accumulated DNA platination products in dorsal root ganglion cells are causative for the functional impairment of peripheral nerve fibers in mice after the application of cisplatin. We, therefore, first measured the kinetics of formation and removal of Pt-(GG) adducts in various nervous tissue cells of three mouse strains differing in their capability to eliminate DNA damage via the NER pathway. Second, we investigated whether the differential DNA repair capacity of NER-proficient and -deficient mice determines the accumulation of Pt-DNA adducts after repetitive exposure to cisplatin and whether this, in turn, correlates with the severity of peripheral neuropathy.
Materials and Methods
Animals. C57BL/6 mice were used as DNA repair-competent wild-type (WT) mice throughout all experiments. Syngenic Xeroderma pigmentosum complementation group A (XPA)-deficient mice were a generous gift from Dr. K. Tanaka (University of Osaka, Osaka, Japan). Both strains were propagated by house breeding and XPA–/– homozygocity was verified by PCR analysis (Nakane et al., 1995Study design. For the detection and quantification of specific DNA platination products in nervous tissue cells and for the establishment of DNA repair kinetics, mice were injected with a single dose of cisplatin (2 or 10 mg/kg body weight, i.p.; Platinex, Bristol, München). Animals were killed at various time points between 6 h and 5 d after cisplatin administration. For the measurement of cisplatin-DNA adducts, dorsal root ganglia (lumbar) and lumbar enlargement of the spinal cord were removed on ice and frozen immediately in liquid N2.
In the second set of experiments male WT and XPA mice were exposed to repetitive applications of cisplatin. Starting at the age of 12 weeks, the animals were injected with 0.5 mg of cisplatin/kg body weight twice per week up to cumulative doses of 32 mg (WT) or 8 mg (XPA). To prevent renal damage, all mice were intraperitoneally injected with 1 ml of saline solution immediately after each treatment. In all experiments, control animals were included which were injected with sterile saline.
Groups of 10 randomly selected mice were subjected to electrophysiological examination at the indicated cumulative doses and animals were killed immediately after the examination by supramaximal anesthesia with diethyl ether. Samples of neuronal tissues were removed and stored frozen as above.
Electrophysiological examination of motor and sensory nerve fibers. All electrophysiological examinations were performed under general anesthesia with phenobarbital (inactin, i.p., 50 mg/kg body weight; Sigma, St. Louis, MO). To minimize the influence of the body temperature on the nerve conduction, animals were placed on a water-heated glass plate maintaining the hind paw skin temperature at 37°C.
Sensory- and motor-nerve conduction velocities were calculated using recordings of H- and M-responses after stimulation of sciatic nerves as described previously (Verdu et al., 1999
The compound muscle action potentials elicited by orthodromic conduction (M-response) and by the monosynaptic reflex arc (H-response) were recorded from the fourth interosseous muscle of the hindfoot with a pair of microneedle recording electrodes. After amplification (INH four-channel differential amplifier; World Precision Instruments, Berlin, Germany), the evoked action potentials were sampled and analyzed by means of a CED micro1401 system with the Signal for Windows version 2.05 software (Cambridge Electronic Design, Cambridge, UK).
Peak-to-peak amplitudes of M- and H-responses were measured. The response latencies (in milliseconds) were defined as the interval between the stimulus artifact and the beginning of the M- or H-response. The distance between the two stimulation points was measured over the skin with the hip and knee in a flexed position. Motor-nerve (MNCV) and H-reflex-related sensory-nerve conduction velocity (SNCV) were calculated as follows: MNCV, m/s = distance (sciatic notch to ankle)/latency (sciatic notch M-response – ankle M-response); SNCV, m/s = distance (sciatic notch to ankle)/latency (ankle H-response – sciatic notch H-response).
Mean values for each experimental group were calculated for amplitudes of H- and M-responses as well as for SNVC and MNCV.
Cell-type-specific measurement of cisplatin-induced DNA adducts. Frozen sections (8 µm) were prepared from the collected tissue samples and were placed onto ImmunoSelect adhesion slides (Squarix Biotechnology, Marl, Germany) to prevent cell loss during additional processing. Twenty randomly selected sections of each tissue sample were analyzed by immunostaining.
Histological staining and cell type identification. To allow cell-type-specific quantification of DNA adducts neurons and glial or satellite cells were labeled by immunohistochemical staining. After fixation (methanol, –20°C, 12 h), oligodendrocytes and satellite cells were stained with anti-(CNPase) antibodies (5 µg/ml in PBS, 90 min, 37°C; Sigma) and secondary antibodies labeled with Alexa Fluor 488 (5 µg/ml in 1% casein/PBS; Invitrogen, Eugene, OR). Neurons were labeled with NeuroTrace 530/615 red fluorescent Nissl Stain (Invitrogen) according to the manufacturer's protocol. The nuclear DNA was counterstained with 4,6,diamidino-2-phenylindole (DAPI) (1 µg/ml) and slides were mounted in dithioerythritol (4.4 mg/ml; Sigma). The localization of nuclei (blue) of neurons (red) or glial and satellite cells (green) was recorded by laser-scan microscopy (Axiovert 100/LSM 500; Zeiss, Oberkochen, Germany) and images were stored electronically. The coverslips were carefully removed and the slides were further processed for DNA adduct staining.
Quantification of Pt-(GG) adducts in nuclear DNA by immunocytological assay. Immunofluorescence staining and measurement of specific DNA platination products were performed essentially as described by Liedert et al. (2006)
Immunofluorescence and DNA fluorescence signals were measured by digital image analysis and adduct levels in the nuclear DNA of individual cells were calculated as arbitrary fluorescence units (AFUs) by normalizing integrated antibody-derived fluorescence signals from >400 nuclei/cell type to the corresponding DNA content of the same cell. Statistical significance was assessed by means of ANOVA for repetitive AFU (±SEM) measurements performed using SPSS (Chicago, IL) 9.1 software. A two-sided p value of <0.05 was regarded as indicating statistical significance.
Statistical analysis. One-factorial ANOVAs were performed to compare mean body weights, mean amplitudes of H- and M-responses and mean sensory and motor conduction velocities in the groups of WT mice. One-factorial ANOVAs were used to compare the mean level of Pt-DNA adducts in neurons and glial cells of the spinal cord and neurons and satellite cells of DRG in WT mice (factor "Dose": control vs 4, 8, 16, and 32 mg groups).
One-factorial ANOVAs were performed to compare the same variables between the groups of XPA mice. One-factorial ANOVAs were used to compare the mean level of Pt-DNA adducts in neurons and glial cells of the spinal cord and neurons and satellite cells of DRG in XPA mice (factor Dose: control vs 4 and 8 mg groups).
Post hoc comparisons were performed using multiple t tests with Bonferroni corrections. The level of significance was 0.05.
Results
Visualization and measurement of cisplatin-DNA lesions in distinct cell types of the nervous system
To determine the level of DNA adducts in the nuclear DNA of mouse nervous tissue cells, we established a quantitative immunocytological assay (ICA) by combining cell-type-specific staining techniques and monoclonal antibody-based immunocytological measurement of DNA intrastrand cross-links. After injecting mice with cisplatin, cryosections of DRG and spinal cord (SC) were stained immunohistochemically for CNPase (a marker for glia and satellite cells; green) and by fluorescent Nissl stain for neurons (red), and were counterstained for nuclear DNA with DAPI (blue) (Fig. 1A,B). After recording, the digital images the slides were further processed for quantitative ICA analysis of Pt-(GG) cross-links in DNA using antibody R-C18 (Liedert et al., 2006
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Figure 1. Visualization of Pt-(GG) adducts in the nuclear DNA of defined cell types of mouse dorsal root ganglia. A, B, DRGs were dissected from C57BL/6 mice (untreated or 24 h after i.p. application of 2 mg cisplatin/kg body weight) and cryosections were first stained for cell-type-specific markers (neurons: fluorescent Nissl stain, red, arrows; glial cells: CNPase, green, arrowheads) and counterstained for DNA with DAPI. C, After electronic recording of digital images, the sections were further processed for ICA analysis (see Materials and Methods) and immunostained for Pt-(GG) intrastrand cross-links in the nuclear DNA using monoclonal antibody R-C18 and green fluorescent secondary antibodies.
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Figure 2. Levels of Pt-(GG) DNA adducts in different cells types of the peripheral and CNS of mice after a single dose of cisplatin (2 or 10 mg/kg, i.p.). Cryosections from DRGs or spinal cord tissues were immunostained as in Figure 1 and integrated fluorescence signals from individual nuclei (blue, DNA; green, Pt-[GG]) (see Fig. 1) were measured by fluorescence microscopy-based quantitative image analysis. Adduct-related signals were normalized for the actual DNA content of each cell and expressed as AFUs. Columns represent mean values (±SEM) from each 200 cells. A, Comparison of peak adduct levels 24 h after drug injection. B, Repair kinetics in DRG cells after a dose of 10 mg/kg.
Formation and repair of Pt-(GG) cross-links in neuronal tissue cells of mice proficient or deficient for nucleotide excision repair functions
Next, we determined the DNA adduct kinetics in cells of the peripheral nervous system and CNS of mice after acute exposure to cisplatin (Fig. 3). The Pt-(GG) levels increased slowly in all cell types and peaked 24 h after drug application. Within the next 24–48 h, a major part of the lesions was removed from the nuclei of all four cell types by DNA repair, whereas the rest representing 30–50% of the initial burden remained unrepaired at a plateau level even 5 d after exposure. The persistence of cross-links at that time point was not caused by the exhaustion of cellular DNA repair systems because adduct kinetics at a higher dose of cisplatin (10 mg/kg) resulted in higher peak adduct formation (see above), but displayed nearly unaltered repair characteristics (Fig. 2B).
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Figure 3. Formation and repair of Pt-(GG) intrastrand cross-links in specific nervous tissue cells of mice after a single dose of cisplatin. C57BL wild-type mice and NER-deficient (XPA–/– or XPC–/–) mice were injected with cisplatin (2 mg/kg, i.p.) and killed at different times after application. Levels of Pt-(GG) adducts in the nuclear DNA of specific cell types were measured by ICA analysis as in Figure 1 and 2. Data points represent mean values (±SEM) of 200 evaluated cells randomly selected from tissue sections from two animals per time point and mouse strain.
To illuminate the contribution of a distinct repair mechanism in removing Pt-(GG) intrastrand cross-links from the genomic DNA of primary nervous cells, we analyzed, in parallel, mice with deficiencies in the NER pathway, namely, animals lacking either functional XPA or XPC protein (Fig. 3). As for C57BL/6 WT mice, the DNA adduct levels in cells of XPA–/– mice peaked 24 h after a single dose of cisplatin (2 mg/kg), showing a similar cell-type-specific adduct distribution with high values in satellite cells and neurons of the DRG, and comparatively lower levels in glial cells and neurons of the spinal cord. However, in XPA mice, the application of cisplatin induced up to twofold higher Pt-(GG) adduct levels compared with the corresponding cell type of identically treated WT mice, although the XPA-deficient cells were still competent to remove the intrastrand cross-links between 24 and 48 h at similar repair rates as their WT counterparts. Neuronal cells of XPA–/– mice, too, left about half of the peak adduct burden unrepaired by day 5 after treatment, resulting in about two times higher plateau levels than in WT mice.In XPC-deficient mice treated with the same dose of 2 mg cisplatin/kg, the cell-type-specific adduct levels at 24 h displayed similarly high values as in XPA mice. However, in sharp contrast to the neuronal cells of WT and XPA mice, the cells of XPC mice were completely deprived in repairing Pt-(GG) adducts. Once formed, the lack of functional XPC protein resulted in a long-term persistence of the intrastrand cross-links in all four cell types of the nervous tissue (Fig. 3).
Drug-induced DNA adducts accumulate in dorsal root ganglion and spinal cord cells during chronic treatment with cisplatin
To mimic the clinical situation with repetitive treatment cycles and to prove whether the unrepaired portion of DNA intrastrand cross-links observed after a single application of cisplatin give rise to the accumulation of such lesions during chronic exposure, we injected WT mice with doses of 0.5 mg cisplatin/kg body weight twice per week. When the animals were killed after 2, 4, 8, 16, or 32 weeks of treatment, the ICA measurement indeed revealed a steady increase of Pt-(GG) DNA adduct levels with the cumulative dose in all four cell types (Fig. 4A). Similar to the situation after acute exposure (see above), cells in the dorsal root ganglia carried higher adduct burdens than cells of the spinal cord, and neurons were less affected than glia or satellite cells in the same anatomical location.
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Figure 4. Accumulation of Pt-(GG) intrastrand cross-links in spinal cord and dorsal root ganglion cells of mice after repetitive applications of cisplatin. Syngenic C57BL/6 wild-type or XPA-deficient mice were injected twice weekly with cisplatin (0.5 mg/kg, i.p.) and were killed at various cumulative doses of the drug (wild type, 2–32 mg/kg; XPA, 2–8 mg/kg; 10 animals per dose) 5 d after the last application. DNA adduct levels (mean ± SEM) were measured by ICA analysis in randomly selected tissue sections of DRG or spinal cord samples from different mice as described in Figures 1 and 2.
Accumulation of Pt-(GG) adducts is higher in neuronal cells of mice with dysfunctional nucleotide excision repair
When NER-deficient XPA–/– or XPC–/– mice were exposed to the same repetitive application of cisplatin, increasing mortality was already observed at total doses >10 (XPA) or 6 (XPC) mg/kg body weight. The histopathological inspection of moribund animals revealed morphological alterations in the kidney. Therefore, XPC mice were excluded from this experiment and XPA mice were treated only up to a cumulative dose of 8 mg cisplatin/kg with no indications of renal failure. The cells of the central and peripheral nervous tissues carried, at each time point, significantly (p < 0.05) higher levels of Pt-(GG) adducts compared with the corresponding cell types in NER-proficient WT mice (Fig. 4B). This accelerated accumulation of cisplatin lesions was particularly pronounced at lower cumulative doses of 2 and 4 mg/kg, resulting in adduct burdens that were about twice as high as in WT mice.Mice with reduced DNA repair capacity exhibit earlier onset of neurophysiological alterations during repetitive exposure to cisplatin
Next, we used this animal model to investigate the role of DNA adduct accumulation as a crucial initial event in cisplatin neurotoxicity finally leading to functional impairment. Mice were exposed to cisplatin as above and examined for alterations of electrophysiological parameters typically observed in drug-induced neuropathy. At various cumulative doses, groups of 10 animals were subjected to quantitative neurography. After proximal or distal stimulation of the sciatic nerve in foot muscles, the MNCV and SNCV as well as the amplitudes of the M- and H-responses were measured.In WT mice, the amplitude of the H-responses steadily decreased with increasing cumulative doses (Table 1) (df = 4; F = 5.9; p < 0.001). A significant decrease of H-response amplitudes was observed at total doses of 16 mg of (p = 0.003) and 32 mg cisplatin (p = 0.001). SNCV values in the sciatic nerve also changed with prolonged treatment (df = 4; F = 3.54; p = 0.013) and a significant decrease was observed at a cumulative dose of 32 mg cisplatin (p = 0.031). In contrast, no significant changes were observed for the M-responses and the MNCV compared with the untreated controls.
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Table 1. Electrophysiological changes in the course of repetitive exposure of NER-proficient (WT) and NER-deficient (XPA) mice to cisplatin
Electrophysiological examination of identically treated XPA mice revealed similar unaltered motor nerve values (MNCV and M-amplitude) throughout the dose range (0–8 mg cisplatin/kg). However, significant decreases of H-responses and SNCV values were measurable already at a cumulative dose of 4 mg/kg with an additional reduction at 8 mg/kg (Table 1). At the latter dose a decrease of the H-responses by 61% and of the SNCV by 34% was observed in XPA mice compared with reductions of only 36% and 4% for the respective values in identically exposed WT mice. No significant discrepancies between both strains were observed for all four parameters when comparing the untreated control groups.Inverse correlation between the level of accumulated DNA adducts and the magnitude of electrophysiological changes
Because both syngenic mouse strains only differed in their ability to repair damaged DNA, the electrophysiological data strongly suggested that persisting cisplatin adducts represent the causative molecular trigger for the onset of PNP-like deficiencies. To further support this notion, we correlated the levels of unrepaired Pt-(GG) adducts in DRG neurons at a given cumulative dose of cisplatin to the corresponding values for SNCV and H-amplitude. For both mouse strains, WT and XPA–/–, highly significant inverse correlations were found for the H-responses (WT mice, Spearman's r = –0.94, p = 0.005; XPA mice, r = –0.95, p = 0.05) as well as for the SNCV values (WT mice, r = –0.84, p = 0.036; XPA mice, r = –0.96; p = 0.045), with a steep drop of both parameters at Pt-(GG) cross-link levels between 0.4 and 0.8 AFU values (Fig. 5). A similarly stringent inverse correlation was found for the combined values from both mouse strains and for the persisting adduct levels in DRG satellite cells (H-amplitude, r = –0.86, p = 0.0015; SNCV, r = –0.89; p = 0.0006).
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Figure 5. Levels of unrepaired Pt-(GG) adducts in DRG cells are significantly correlated with the magnitude of electrophysiological alterations. Both the amplitudes of the H-responses and the SNCV measured in NER-proficient (WT) or -deficient (XPA) mice after repetitive applications of cisplatin display significant negative correlations to the proportion of persisting DNA intrastrand cross-links in DRG neurons. Animals were injected twice weekly with cisplatin as described in Figure 4 and electrophysiological examination as well as the measurement of DNA adducts were performed at day 5 after the last application. The inverse correlations were highly significant (p < 0.05) for the H-amplitudes as well as for the SNCV values independently from the repair phenotype of the animals.
Discussion
In this study, we attempted to identify the molecular pathogenic mechanisms that trigger the onset of polyneuropathy frequently observed in cancer patients after repetitive treatment with cisplatin. For this purpose, we have established a mouse model that allowed the concomitant analyses of PNP-related electrophysiological parameters as well as the formation and processing of structurally defined DNA reaction product of cisplatin in individual cells of neuronal tissues.Until now, the measurement of platinum concentrations in tissue samples or in isolated DNA was based predominantly on spectroscopic methods (Reed et al., 1987
When analyzing cryosections of nervous tissues from cisplatin-treated mice with Mab R-C18, we found that the formation of the main intrastrand cross-link, Pt-GG, was about twofold higher in peripheral DRG neurons and satellite cells than in the corresponding cells of the spinal cord (Fig. 2). Most likely, this is because of the protective role of the blood–brain barrier, which shields brain and spinal cord cells from exposure after systemic application of cisplatin (Gregg et al., 1992
Inefficient DNA repair does not account for the higher adduct levels in satellite or glia cells because their adduct kinetics revealed similar repair profiles to the adjacent neurons (Fig. 3). Therefore, increased uptake (Safaei and Howell, 2005
Despite being repair-proficient for Pt-(GG) adducts, cells of the CNS and PNS were incapable to completely remove these lesions from their DNA. As shown for cells of other mouse tissues like liver or kidney (Liedert et al., 2006
Previous observations in various experimental systems suggested a general downregulation in terminally differentiated neuronal cells of several DNA repair mechanisms, including global genomic NER (Nouspikel and Hanawalt, 2002
30% within 4 d. Although a previous study with siRNA-mediated downregulation of XPA in cisplatin-exposed cell lines suggested that recombination repair functions might be able to partly substitute for impaired NER (Cummings et al., 2006
Together, our observations suggest a new model of how reaction products of cisplatin with nuclear DNA are processed in mouse cells: both the rapidly formed monoadducts as well as the slowly occurring secondary (GG) intrastrand cross-links are efficiently recognized and repaired by intact NER machinery. After the loss of functional XPA or XPC proteins, guanine monoadducts are no longer removed, resulting in a significantly higher formation of Pt-(GG) lesions. Those cross-links can still be eliminated to some extend in cells lacking functional XPA, but are completely persistent in XPC-deficient cells. Interestingly, in the rare cases of adult patients with inherited XPC defects, this inability of the NER system was associated with significant neuronal cell loss in DRGs, most likely because of accumulated DNA damage from endogenous metabolites (Robbins et al., 2002
Thus, we had available an animal model where after application of the same dose of cisplatin, the mouse strains differed in the amount of unrepaired intrastrand cross-links in peripheral and central nervous tissue cells. We used WT and XPA mice to investigate whether repetitive drug exposure resulted in strain-specific differential accumulation of DNA damage in those cells and whether this, in turn, correlated to alterations in electrophysiological parameters typically observed in cisplatin-induced neuropathy. Derogations in the sensory response of the tibial nerve were shown to be reliable indicators of cisplatin neurotoxicity in mice and rats (De Koning et al., 1987
During low-dose chronic exposure to cisplatin, we indeed found for both mouse strains a progressive accumulation of Pt-(GG) lesions in DRG cells as well as a reduction of H-response amplitudes and a slowdown of the SNCV, indicating a progressive sensory neuropathy. Furthermore, we could show, separately for each mouse strain and in a cumulative data analysis, a significant correlation between the levels of persisting DNA adducts and the severity of functional sensory impairment (Table 1, Fig. 5). Although satellite cells accumulate significantly higher adduct levels than neurons after acute or chronic exposure, additional subcellular analyses in DRG tissue are needed to rule out whether cisplatin induces predominantly a neuropathy rather than a Schwann cell pathology in mice.
Our results clearly demonstrate that suboptimal DNA repair in critical cells of the nervous system accelerates the accumulation of DNA cross-links during chronic application of cisplatin and may thus represent an important risk factor for drug-induced neurotoxicity. Because humans vary up to 10-fold in their individual DNA repair capacity of distinct cell types as shown (e.g., for blood lymphocytes) (Buschfort-Papewalis et al., 2002
Footnotes
Received Feb. 6, 2007; revised June 26, 2007; accepted July 5, 2007.This work was supported in part by Deutsche Forschungsgemeinschaft Grant FG 236 TP 1a (J.T.) and a grant of the Medical Faculty, University Duisburg-Essen (IFORES Program) (V.L., J.T.). We thank Dr. Kiyoji Tanaka (University of Osaka, Osaka, Japan) and Dr. Leon Mullenders (University of Leiden, Leiden, The Netherlands) for providing the XPA and XPC mice, respectively, and Beate Karow for expert technical assistance.
Correspondence should be addressed to Dr. Juergen Thomale, Institute of Cell Biology, Cancer Research, University of Duisburg-Essen Medical School, Hufelandstrasse 55, D-45122 Essen, Germany. Email: Juergen.thomale{at}uni-essen.de
Copyright © 2007 Society for Neuroscience 0270-6474/07/279451-07$15.00/0
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