1 Radiobiological Institute, University of Munich, Schillerstrasse 42, D-80336 Munich, Germany and
2 Institute of Molecular Radiation Biology, GSFNational Research Centre for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany
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Abstract |
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Abbreviations: mtDNA, mitochondrial DNA; MSI, microsatellite instability; ROS, reactive oxygen species
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Introduction |
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Mutations in the mitochondrial genome can give rise to maternally inherited or spontaneous mitochondrial disorders (5). mtDNA aberrations have also been reported for solid tumours and haematological malignancies (3,68). However, it is as yet unclear, how an mtDNA variant genome expands and replaces part of (heteroplasmy) or all (homoplasmy) wild-type mtDNA in the affected cells. Observations from pedigrees of families carrying the mitochondria syndrome MELAS (mitochondria encephalopathy, lactic acidosis and stroke-like episodes) and cells in vitro from MELAS sufferers, indicate a tendency for homoplasmy (9,10).
As a result of the proximity of mtDNA to reactive oxygen species (ROS) produced in the respiratory chain, higher levels of oxidative damage have been reported in mtDNA compared with levels in nuclear DNA (11). Despite the evidence for base excision repair, direct damage reversal, mismatch repair and recombinational repairhowever, not for nucleotide excision repair (12)the mutation rate in mtDNA is 10-fold higher than in nuclear DNA (13). The accumulation of damage in mtDNA with time (14,15), supports the free radical theory of aging proposed earlier (16), which suggests a link between aging, mitochondrial deterioration and accumulation of oxidative damage.
Exposure of cells to ionizing radiation results in oxidative damage of DNA bases, but also DNAprotein cross-links and single- and double-strand breaks. The response to the DNA damage and the efficacy of DNA repair will determine the fate of the irradiated cell. However, the progeny of survivors of irradiated cells exhibit long-lasting consequences like delayed mutation, clonal heterogeneity, delayed cell death and persisting genomic instability (17). These biological endpoints have been correlated with the perseverance of oxidative stress (18). The resulting mutations in nuclear DNA could predispose the cell to tumour transformation and also lead to a characteristic spectrum of mutations in mtDNA.
Following the interaction of DNA and ROS, generated from endogenous sources or after exposure with ionizing radiation, the most abundantly damaged base is 8-oxodG (19). Although, with a frequency of <5%, this lesion can give rise to G:C T:A transversions. ROS also challenge the dNTP pool causing 8-oxodGTP, which after incorporation opposite dC or dA by DNA polymerases can generate A:T
C:G transversions during the next round of replication (20).
Following the accident at the nuclear power plant in Chernobyl in 1986, the population living in the region was exposed to radioactive isotopes from the fall out. A few years after the accident, a dramatic increase in thyroid tumours in children was diagnosed (21) in the areas most contaminated by radioactive iodine (22). These tumours were characterized by chromosomal instability and RET proto-oncogene rearrangements, which were thought to be the result of double-strand breaks induced by ionizing radiation (2327). We have shown recently, that microsatellite instability (MSI) was virtually absent in the majority of radiation-associated thyroid tumours from Belarus, with the exception of aggressive, fast growing thyroid tumours emerging <9 years after the accident. However, MSI was a feature of spontaneous thyroid tumours from Munich, which had no radiation history (28,29).
If the exposure of thyroid cells to ionizing radiation causes sufficient genetic disruption to increase the risk of tumourigenenesis, then radiation damage to mtDNA could also increase the possibility of mitochondrial mutations. After investigating the stability of mtDNA in 126 thyroid tumours from Belarus, however, no indication of an increased mutation frequency in mtDNA was detected. This was in contrast to a high frequency of mtDNA alterations in 40 spontaneous thyroid tumours from Munich. The analysis of all tumours irrespective of their provenance revealed that the age of the patients at the time of surgery correlated with the mutation frequency of mtDNA.
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Materials and methods |
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Samples from tumourous and non-tumourous tissues were also obtained from 40 thyroid cancer patients (mean age at surgery 53.9 ± 15.3 years) with no radiation history from the Martha-Maria Hospital, Munich. The specimens comprised of 16 papillary carcinomas (40%), seven follicular carcinomas, five solid adenomas, three medullar carcinomas, two undifferentiated carcinomas, two carcinomas without specified histoarchitecture, one follicular adenoma, one papillary adenoma and three specimens without classification.
Genomic DNA was extracted from 20 mg of each tumourous and non-tumourous tissue using a DNA isolation kit (Qiagen, Hilden, Germany).
Analysis of mtDNA
Tumourous and non-tumourous samples from 126 patients from Belarus and 40 patients from Munich were used to investigate MSI in mtDNA. Instability of mitochondrial microsatellites was determined by PCR amplification of the mito-s fragment (accession no. NC_001807.2, positions 73337; 2), using primer mito-s-fwd (ACA GGC GAA CAT ACT TAC TAA AGT G, positions 181205) and mito-s-rev (GTT TGG CAG AGA TGT GTT TAA GTG CTG, positions 353327). Primer mito-s-fwd was 5'-labelled with fluorescein by the manufacturer (Life Technologies, Karlsruhe, Germany).
DNA amplification was performed in a total volume of 10 µl containing 1020 ng DNA template in 10 mM TrisHCl (pH 8), 50 mM KCl, 1.5 mM MgCl2, 100 µM each of dNTPs, 0.5 U Taq-DNA polymerase (Life Technologies) and 0.3 nmol of each primer. PCR was based on 36 cycles of 94°C for 45 s, 53°C for 1 min 15 s and 71°C for 45 s with an initial denaturation step of 94°C for 4 min and a final extension at 71°C for 7 min. PCR products were mixed 1:1 with loading buffer (de-ionized formamide, 0.2% dextran blue), denatured at 95°C for 3 min and electrophoresed through a 6% denaturing polyacrylamide gel (Sequagel XR, National Diagnostics, Hull, UK) using an ALF automated sequencer (Amersham Pharmacia, Freiburg, Germany).
Mito-s fragment sizes from all 166 patients were determined by Fragment Analysis Software (Amersham Pharmacia). The differences between fragment length from tumourous and non-tumourous tissue were analysed using SigmaPlot 2000 statistics program module. Values close or outside the 99% prediction interval were considered for sequencing.
Sequencing of mito-s fragment
The mito-s fragment from DNA samples form tumourous and non-tumourous tissue from 25 thyroid-cancer patients from Belarus, 12 patients from Munich and from 20 healthy blood donors, were sequenced. Sequences of the hypervariable region II from mitochondria were pre-amplified using mito-p-fwd primer (TCA CCC TAT TAA CCA CTC, positions 1431) and mito-p-rev (TTG ATG AGA TTA GTA GTA TGG, positions 488468). The resulting fragment was called mito-p. PCR conditions were identical as described above, with the exception of the annealing temperature of 59°C and a total volume of 30 µl. PCR fragments were purified using a PCR purification kit (Qiagen, Hildesheim, Germany) and checked by 2% agarose gel electrophoresis.
Approximately 100 fmol of mito-p fragment were used as template for the cycle-sequencing reaction using the CEQ DTCS kit (Beckman Instruments, Munich, Germany). The sequencing reaction was primed using 3.2 pmol of mito-fwd-seq oligomer (AGC ATT GCA AGA CGC TGG AGC CGG AGC; positions 84110) following the manufacturers instructions. DNA amplification was based on 30 cycles of 96°C for 20 s, 59°C for 20 s and 60°C for 4 min preceded by an initial denaturation step at 94°C for 30 s and terminated by a final extension at 71°C for 4 min. PCR products were ethanol precipitated, washed twice with cold 70% ethanol, dried in a speed vac for 40 min and re-dissolved in sample loading solution provided by the manufacturer. Samples were transferred to Beckman 96-well plates and loaded into a CEQ2000 automated sequencer (Beckman). mtDNA sequences were analysed using Genomax Vector NT3 software (InforMax, Oxford, UK), software provided by the HGMP Resource Centre (Cambridge, UK) and by HUSAR (German Cancer Research Centre, Heidelberg, Germany).
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Results |
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Comparisons of the mito-s sequences from tumourous and non-tumourous DNA samples from these eight patients, revealed that mutations were exclusively found in a microsatellite based on a poly-cytidine sequence (positions 303315 of the Cambridge notation), interrupted by a single T.
In contrast to the published Cambridge Reference sequence, the 3'-C-repeat of the poly-cytidine microsatellite of all our samples contained six cytosines and not five as the database suggests (hence C7TC6). This discrepancy was considered a mistake in the database. All mutations found in the C7TC6 microsatellite are summarized in Table I. The thyroid tumours from two patients from Belarus (S323 and S348), each showed deletion of one cytosine of the 5'-C-repeat, whereas most thyroid tumours from patients from Munich showed insertion of one cytosine (with the exception of S105). Only in two tumours from Munich (S10 and S137) the insertion of one cytosine into the 3'-C-repeat was found, remarkably in tumours that had shown increased MSI in an earlier investigation (two, respectively, four altered nuclear microsatellites of 26 markers tested; 29).
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The mean age of the patients from Belarus was 16.4 ± 8.7 years and the mean age of the patients from Munich was 53.9 ± 15.3 years. Hence, the difference in the frequency of mutational events in the mito-s sequence between the two patient collectives could be the result of the age of the patients. Therefore, the patients were pooled according to their age at surgery into intervals of 10 years and the average frequency of C7TC6 microsatellite mutations in each interval was calculated. The result was a seemingly steady increase in the frequency of C7TC6 microsatellite mutations in thyroid tumours with the age of the patients (Figure 1, filled columns, scaling left ordinate). The number of patients per interval (step line, scaling right ordinate) varied strongly, because of the high number of thyroid tumours from children from Belarus.
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All sequences were aligned and the consensus sequence became the basis of further analysis. Two differences between the mito-s consensus and the Cambridge Reference sequences became immediately apparent: at position 83 bp of the mito-s consensus sequence, the Cambridge Reference sequence defined adenine, whereas in all samples (including control DNA from healthy donors) guanine was identified. A second difference confirmed results described above: an insertion of cytosine into the C7TC5 microsatellite (Cambridge sequence) in position 138 bp of the mito-s consensus sequence, was present in all samples to yield the C7TC6 microsatellite.
Polymorphisms in the mito-s sequences of non-tumourigenic tissue (and healthy controls) were grouped into haplotypes and listed in Table II. The consensus sequence and positions of polymorphisms are given in the top of Table II
. Positions of insertions of bases into the Cambridge Reference sequence are indicated by x. The position of the C7TC6 microsatellite (position 123137) is indicated.
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Discussion |
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Differences in the mito-s fragment size of 1 bp between tumourous and non-tumourous samples were found in two out of 126 (or 1.6%) patients from Belarus, and in four out of 40 thyroid tumours from Munich, where in another two tumours 2 bp differences were identified (total of 15%, see Table I). All these mutations were limited to the C7TC6 microsatellite. Sequence data from these eight and from a further group of 29 thyroid-cancer patients, showed that none of the tumours harboured base substitutions or combinations of deletions and insertions elsewhere in the mito-s fragment. This eliminated the possibility that mutations, which would not have caused size differences, would have gone undetected, and thus would have entailed an underestimation of mutational events.
In an earlier investigation, we found that a nearly identical set of thyroid tumours from Belarus was characterized by stability of nuclear microsatellites (with the exception of a small group of early and aggressive tumours) and that many thyroid tumours from Munich showed MSI (two or more alterations of 26 microsatellites tested). Notably, the two tumours from Munich with two mutations in the mitochondrial C7TC6 microsatellite also showed instability of nuclear microsatellites. However, in another eight thyroid tumours from Munich with MSI we found no difference in mito-s fragment size.
The occurrence of two mutations in the mitochondrial C7TC6 microsatellite in 126 thyroid tumours from Belarus was significantly different to the eight mutations found in the 40 thyroid tumours from Munich (P = 0.0002). This could be the result of the mean age of the two collectives of patients at surgery. A correlation of the average C7TC6 mutation frequency in thyroid tumours with the age of the patients at surgery was identified (Figure 1). The slope of the linear regression line gave 0.22 mutations in the mito-s fragment per 50 years or a mutation frequency of 2.5x10-5/bp/year. This mutation frequency was similar to previously published data, which were based on the inclusion of the C7TC6 microsatellite (31). Exclusion of the C7TC6 microsatellite reduced the mutation frequency of the D-loop ~1000-fold (32,33).
In addition to the mutations found in the C7TC6 microsatellite, the sequencing of the mito-s fragment of 37 thyroid tumour patients revealed a number of polymorphisms. The average frequency of polymorphisms for the patients from Belarus was slightly elevated compared with patients from Munich. However, the ratio of mutations within the C7TC6 microsatellite and the polymorphisms in the rest of the mito-s fragment was 0.88 for patients from Belarus and 1.60 for the patients from Munich (healthy controls 0.92; see Table III). This over-representation of microsatellite mutations in thyroid tumours from Munich could be a result of a higher number of cell divisions and the accumulation of replication errors before the onset of tumourigenesis. Hence, the increased number of altered microsatellites found in sporadic thyroid tumours from Munich would reflect the accumulation of mutations in an aged thyroid stem cell and would be proof for the clonality of the tumour rather than a diagnostic marker. The scarcity of C7TC6 microsatellite mutations found in thyroid tumours from Belarus would then be a strong indication that a mutator phenotype with MSI would not necessarily be a prerequisite for the process of tumourigenesis.
There remained the possibility that (some of) the polymorphisms could be the result of somatic mutations in thyroid stem cells during early development (see Table II). These mutations could have been the result of oxidative damage by ROS following the exposure to radioactive iodine or as a result of cellular respiration (16,34). The spectrum of base substitutions generated by 8-oxo-guanine would predominantly cause G:C
T:A transversions, whereas ROS attack on the nucleotide pool would yield 8-oxo-GTP, which could result in A:T
C:G transversions (20,3537). However, none of the polymorphisms found in thyroid-cancer patients showed evidence of such mutations.
The absence of the spectrum of mutations potentially generated by ROS activity is in contrast with the hypothesis of persisting oxidative stress following exposure to ionizing radiation (18). The results presented here indicate that radiation-induced and persisting oxidative stress is limited to nuclear DNA and either the higher levels of oxidative stress protects mitochondria from radiation-induced oxidative stress, or some selection against mitochondria with damaged genomes prevent the fixation of mutations in mtDNA. An indication for such mechanisms can be taken from reports of higher proportion of cells with elevated levels of oxidative stress entering apoptosis or necrosis compared with cells with normal levels (38). Thus, persisting oxidative stress following the exposure to radioactive iodine in the thyroid tumour stem cell compartment could lead to reduced fitness of mitochondrial genomes and their elimination from the population.
Our earlier publication which reported an increased instability of nuclear microsatellites in spontaneous thyroid tumours from elderly patients from Munich, compared with the stability of microsatellites from radiation-induced thyroid tumours from children and young adults from Belarus, were in accordance with the frequency of mutations in mitochondrial microsatellites correlating with the age of the patients at surgery presented here (29). As most tumours are considered to be clonal expansions of a single cell, microsatellite variations reflect the genetic status at the start of the expansion process. However, the genetic distance between the tumourous and the non-tumourous tissue is of no diagnostic use, if genetic alterations have accumulated during the life span of stem cells prior to the onset of tumourigenesis. Genetic alterations could only serve as markers for tumourigenesis if it can be shown that they have been shaped by the process of mutation, selection and apoptosis of a progeny of a tumour stem cell. Here it could be shown, that alterations of mitochondrial microsatellites were age-related genetic events, and would not qualify as markers for tumourigenesis.
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Notes |
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Acknowledgments |
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References |
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