1 Radiobiological Institute, University of Munich (LMU), Schillerstraße 42, 80336 Munich, Germany and
2 Institute of Radiobiology, GSF-National Research Center for Environment and Health, Ingolstaedter Landstrasse 1, 85758 Neuherberg, Germany
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Abstract |
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Abbreviations: APC, adenomatous polyposis coli; BRCA1/BRCA2, breast cancer genes 1 and 2; DCC, deleted colorectal cancer; FES, feline sarcoma; hMLH1, human MutL-homolog 1; HNPCC, hereditary non-polyposis colorectal cancer; HPRT, hypoxanthine phosphoribosyltransferase; LOH, loss of heterozygosity; MSI, microsatellite instability; MYC, avian myelocytomatosis; RET, receptor-typ tyrosine kinase; RETINT5, receptor-typ tyrosine kinase, intron V; XRCC1 and XRCC5, X-ray repair cross complementing 1 and 5.
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Introduction |
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Human cancer has frequently been associated with genetic instability. In the majority of human tumours genome instability was characterized as chromosomal instability (aneuploidy, deletions, amplifications) (5). Only a minority of tumours exhibited mutations on a smaller scale, such as nucleotide instability (6), minisatellite instability (7) or microsatellite instability (MSI) (8), the latter manifested by variations in the copy number of the tandem repeat sequences. Instability of microsatellite sequences was most pronounced in cells from patients with hereditary non-polyposis colon cancer (HNPCC) and is attributed to the loss of mismatch repair genes (9). However, various types of sporadic cancers displayed MSI without evidence of defects in mismatch repair, and thus additional mechanisms must be operative (10).
The analysis of microsatellites also provides information on allelic loss in tumours [loss of heterozygosity (LOH)]. Frequently deleted chromosomal regions suggest the presence of tumour suppressor genes involved in the carcinogenic process. Such allelic loss may occur by mechanisms of hemizygous deletion, chromosomal non-disjunction, mitotic recombination or gene conversion.
Recently Soares et al. (11) investigated 46 benign and malignant thyroid tumours without radiation history at eight loci, mapping to four different chromosomes, and found alterations in three tumours in three and more microsatellite markers. Nikiforov et al. (12), investigating 27 microsatellite markers in 17 samples of tumour and non-tumour DNA of post-Chernobyl pediatric thyroid carcinomas, also found no correlation between MSI and thyroid carcinogenesis. However, minisatellite instability was found in three out of 17 tumours compared with none in 20 sporadic thyroid cancers from patients with no history of radiation exposure. An increased frequency of radiation-associated alterations in minisatellite sequences was described earlier by Dubrova et al. (13) investigating germline mutation of minisatellite loci from children born in highly radiation-contaminated areas after the Chernobyl accident. Compared with a British control group, the mutation rate of Belarussian children was increased up to 2-fold, indicating the impact of radiation exposure on germline mutation rate (13).
Here we present a study of 129 patients who developed thyroid tumours following the exposure to radioactive fallout from the Chernobyl reactor accident. We analysed 28 microsatellite markers situated in the vicinity of DNA repair genes, or genes involved in tumorigenesis, or associated with regions of chromosomal breakpoints, previously determined in post-Chernobyl thyroid cancers (14). We identified 11 tumours of Belarussian children (up to 18 years of age) with alterations in more than two microsatellites, 80% of which were LOHs. In a control group of 20 spontaneous thyroid tumours without radiation history (mean age at surgery 56 ± 13 years) from Munich, we found no alterations in the 28 microsatellite loci investigated.
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Materials and methods |
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For comparison we included in our study a group of 10 female and 10 male thyroid cancer patients (mean age at surgery 56 ± 13 years) from Munich (Martha-Maria Hospital) without radiation history (six papillary carcinomas, six folliculary carcinomas, two medullary carcinomas, two undifferentiated carcinomas, one thyroid carcinoma and three carcinomas without specified histoarchitecture).
Tissue material
Fresh tissue samples were immediately snap frozen in liquid nitrogen and stored at 70°C until use. From most cases, formalin-fixed and paraffin-embedded tissue sections were obtained additionally. Pathological findings according to the TNM classification (15) were provided by the Thyroid Cancer Center, Minsk and the Martha-Maria Hospital, Munich. Paraffin-embedded tissue sections were used for histopathological re-classification.
Isolation of genomic DNA and PCR
Up to 20 mg each of tumour and corresponding non-tumorous tissue was cut, and genomic DNA was extracted using a DNA isolation kit (Qiagen, Hilden, Germany). Extracted DNA samples were employed as templates in PCR experiments in order to amplify microsatellite marker sequence. Oligonucleotide primers corresponding to microsatellite loci were purchased from MWG (Ebersberg, Germany). PCR was performed in 10 µl volumes of a mixture containing 10 mM TrisHCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 100 µM dCTP, dTTP and dGTP, 50 µM dATP, 0,7 µCi [-33P]dATP, 0.4 µM each primer, 0.75 U Taq DNA polymerase and 3050 ng DNA. Thirty-six cycles of 94°C for 1 min, 5560°C for 1 min and 71°C for 1.5 min were performed, with an initial denaturation step of 94°C for 2 min and a final extension step of 71°C for 7 min using a Perkin-Elmer 9600 GeneAMp PCR System. PCR reaction products were diluted 2:1 with loading buffer [98% formamide, 0.1% xylene cyanol FF, 0.1% bromophenol blue and 10 mM EDTA (pH 8.0)] and denatured for 5 min at 95°C. Subsequently, 4 µl of this solution were electrophoresed through a 6% polyacrylamide gel containing 7 M urea and 32.5% formamide for 23 h at 50 W. Following electrophoresis, gels were fixed in 10% acetic acid, washed, dried and exposed to X-ray film for 1272 h.
MSI and LOH
MSI was defined as a shift of specific allele bands in the autoradiograph. LOH was defined as a total loss or at least 70% reduction of signal density of one allele in the autoradiograph.
The 28 polymorphic markers were located in or near regions of candidate genes (18 markers) or of chromosomal breakpoints (10 markers) previously determined in post-Chernobyl thyroid cancers (14). As shown in Table I most microsatellite markers were highly polymorphic dinucleotide (CA)n repeats.
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Results |
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Genomic instability in tumour samples
Instability of one or more microsatellite markers was found in 40 matched pairs (31%) of 129 DNA samples from non-tumorous and tumorous tissues from Belarussian thyroid cancer patients. The group consists of mainly female patients of young age. Of all alterations observed, 80% were classsified as LOHs and were found exclusively in tumour DNA. The remaining 20% were alterations of the copy number of the microsatellite repeat sequences, as deduced from modifications of the migration behaviour of the PCR amplified DNA fragments. Representative autoradiographs are shown in Figure 1.
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Amongst the 40 matched DNA samples we identified a set of 11 samples (8.5% of the total 129) exhibiting alterations in at least two markers (Table II). Only 15 microsatellite markers out of all 23 unstable loci were involved in alterations identified in this group of patients. The loci with the most frequently observed variations were hypoxanthine phosphoribosyltransferase (HPRT; altered in four tumours), feline sarcoma (FES), adenomatous polyposis coli (APC), BRCA2, deleted colorectal cancer (DCC), hMLH1, XRCC5 and D10S1675 (each altered in three tumours). Taking patient data such as age and gender into consideration, we observed that particularly the tumours of young, female patients (
18 years of age at surgery) showed the highest rate of microsatellite alterations (up to seven altered microsatellites).
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Discussion |
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In this present study, 40 (31%) thyroid tumours out of 129 showed alterations in at least one microsatellite marker, most of which were LOH. These results correspond to earlier findings of loss of genetic complexity during the development of various other tumours (1720). Within this group of patients we identified a subgroup of 11 (8.5%) patients with two or more altered microsatellite markers. The latter group of patients consists of 10 females and one male with an age at exposure between 1 and 9 years, who developed thyroid carcinoma within 9 years after exposure (age at surgery 13 ± 3 years). This group is somewhat larger than could be expected if microsatellite instability occurred randomly amongst all tumours. It is particularly striking, that within this group there were five patients with four or more alterations. However, each tumour is characterized by a different set of unstable microsatellite markers. Hence, none of the 15 microsatellite markers identified in this subgroup with at least two microsatellite alterations can be used as a `biomarker' for thyroid carcinogenesis.
HPRT, located in the region of the housekeeping gene hypoxanthine phosphoribosyl-transferase, was the most frequently altered microsatellite marker and expressed only LOH. HPRT is one of the three enzymes involved in the synthesis of inositol-monophosphate, a precursor of purine biosynthesis, and has been associated with intestinal and breast carcinomas. It has been suggested that the loss of one allele could result in a perturbation of the dNTP metabolism, leading to early disturbances in DNA transactions (21).
Recent studies have indicated that alterations in microsatellite DNA tend to be associated with proto-oncogenes or tumour suppressor genes, and such alterations at microsatellite loci appeared to play an important role in the development of human cancers (2224). The inactivation of tumour suppressor genes most commonly occurs via mutation of one allele followed by loss of the remaining wild-type allele, or its replacement by a duplicated copy of the mutant allele. In our subgroup of 11 patients with two or more altered microsatellite markers, a high frequency of LOH was observed in oncogenes [FES, avian myelocytomatosis (MYC)] and tumour suppressor genes (APC, BRCA2, DCC). The marker D10S1675, which maps to a region of chromosomal breakpoint 10q26 described earlier (14), also showed a prevalence of LOH. Zedenius et al. (25) and Nikiforov et al. (12) had also detected LOH on 10q (location of markers not defined) in thyroid tumours, indicating the location of a tumour suppressor gene in this region.
In earlier studies, Smida et al. (26) found RET-rearrangements in 65% of papillary thyroid carcinomas of Belarussian children. In our study, we found MSI of RETINT5, which is located in intron 5 of the RET proto-oncogene, in four tumours out of the subgroup of 11 patients with two or more altered microsatellite markers. So far only patient 213 has been included in both studies. Smida et al. (26) detected no RET rearrangement in patient 213; however, an increased level of RET-TK transcript was detected. In this present study, we describe a mutation in the RETINT5 marker sequence in one allele of the tumour sample. Further investigations would be necessary to explore the possibility of a link between the increased transcription of the RET proto-oncogene and the mutation of the microsatellite marker in intron 5.
DNA mismatch repair mechanisms are important factors in the maintenance of genomic stability, and defects in mismatch repair genes were linked to HNPCC development (27). Several genes required for mismatch repair in eukaryotic cells have been identified. We investigated one microsatellite marker, located in the region of hMLH1, which showed LOH in seven tumours (three of the 11 patients subgroup). However, the loss of one allele of hMLH1 was not accompanied by an increased frequency of microsatellite mutations. We concluded that the loss of one allele of hMLH1 did not result in the MSI phenotype and seemed not to be involved in the development of thyroid cancer.
We did not detect any alteration in the 28 microsatellite markers investigated in our reference group of 20 spontaneous tumours. The group involved adult patients (mean age at surgery 56 ± 13 years) without radiation history, with tumours of different types and stages. Considering that 31% of Belarussian cancer patients expressed alterations in at least one microsatellite marker, we expected alterations in six tumours of the reference group. The lack of unstable microsatellites in this reference group was significant (Fisher's exact test; P = 0.0019) and might indicate an induction of genome instability following the exposure to radioactive iodine in the patients from Belarus. Considering the small number of patients without radiation history in comparison with the high number of patients exposed to radiation we can only suggest a link between the exposure to radiation and thyroid carcinogenesis.
Taking together our findings of frequent LOH in tumours from Belarussian patients, but no observed genetic alterations in spontaneous tumours, we conclude that exposure to ionizing radiation initiated genetic instability, which in turn favoured a process of carcinogenesis. Our results also show that young girls in particular were at risk of the induction of thyroid carcinomas following exposure to ionizing radiation. We confirmed previous findings of little importance of MSI in thyroid tumorigenesis in general. However, amongst these patients there is a subgroup of children (mainly girls), whose tumours show signs of genetic instability.
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Acknowledgments |
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Notes |
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References |
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