Mutagenesis, Vol. 16, No. 6, 547-550,
November 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Polymorphic insertion of additional repeat within an area of direct 8 bp tandem repeats in the 5'-untranslated region of the p53R2 gene and cancer risk
Department of Biosciences, Karolinska Institute, Novum, 141 57 Huddinge, Sweden
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Abstract |
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p53R2 is a recently cloned gene that functions in p53-induced DNA repair. In the 5'-untranslated region of the p53R2 gene two direct tandem 8 bp repeats are located. Within the region of these 8 bp direct repeats we have detected the insertion of an additional repeat. In order to determine a possible association of this novel polymorphism with any cancer or population, we carried out genotyping of 843 European and Asian controls and patients with various cancer types. In addition, 26 cancer cell lines were included in the study. No significant difference in polymorphic frequency could be demonstrated for any of the cancer types, although the allelic frequency in melanoma patients was lower than in controls (
2 = 3.28; P = 0.07; OR = 0.32; 95% CI 0.07–1.26). A significantly higher frequency of the polymorphism was detected in the compiled Caucasian individuals compared with Asians (2 = 9.19; P = 0.002; OR = 3.13; 95% CI 1.39–7.43). In one tumour cell line we observed two extra inserted copies of the 8 bp repeat. The functional effect of the insertion polymorphism on the p53R2 gene transcription remains to be determined. |
Introduction |
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The recently identified p53R2 gene, the p53-inducible ribonucleotide reductase small subunit 2 homologue, is part of the p53 pathway (Tanaka et al., 2000
). The tumour suppressive activities exerted by p53 occur mainly through cell cycle arrest, apoptosis and DNA repair (Ko and Prives, 1996). Several target genes of p53, such as p21Waf1 involved in cell cycle arrest and Bax, PIG and p53AIP1 acting in mediation of apoptosis, have been identified (Miyashita and Reed, 1995; Polyak et al., 1997; Oda et al., 2000). The p53R2 gene is one of the identified target genes that has been shown to function in p53-induced DNA repair. It shares about 80% homology with the small subunit (R2) of human ribonucleotide reductase, an enzyme that provides precursors for DNA synthesis and that functions in DNA repair (Jordan and Reichard, 1998). The p53R2 gene is induced by diverse stress signals activating p53, such as DNA damaging agents and p14ARF (Nakano et al., 2000). In response to genotoxic stress, inhibition of p53R2 in cells with an intact p53-dependent DNA damage checkpoint has been found to reduce ribonucleotide reductase activity, DNA repair and cell survival. p53R2 has been shown to accumulate in the nucleus after exposure to genotoxic stress, in contrast to other R2 subunits, which are located in the cytoplasm and mainly function in the supply of dNTPs (Engstrom and Rozell, 1988). The fact that p53R2 is induced by DNA damage and its homology with the small subunit of ribonucleotide reductase indicate an important role of p53R2 in p53-induced DNA repair. Although mutations have not been reported in other p53 target genes, the p53R2 gene differs from other identified p53 targets in terms of its likely involvement in DNA repair, making it a candidate of interest to study for alterations with possible functional consequences. Other DNA repair genes, such as those acting in the mismatch repair are found mutated in some cancers (Lengauer et al., 1998).
In the present study, we detected a novel 8 bp insertion in the 5'–untranslated region (UTR) of the human p53R2 gene, in tandem with two pre-existing repeats (Figure 1). The frequency of the insertion polymorphism was determined in different populations; we genotyped in total 843 individuals that included healthy controls and cancer patients. We also analysed 26 different cancer cell lines. This is one of the first alterations to be reported in the recently cloned gene. No extensive mutational analyses of the p53R2 gene have been reported.
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Materials and methods |
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Study population and DNA extraction
The study populations consisted of 843 individuals that included 81 healthy individuals from Nordic countries, 90 healthy Polish controls, 103 healthy Italian controls, 47 healthy Japanese controls, 49 healthy Chinese controls, 60 sporadic melanoma cases from Nordic countries, 100 lung cancer cases from Nordic countries and 44 lung cancer cases from Hungary, 217 breast cancer cases from Nordic countries, 21 Chinese esophageal squamous cell carcinoma cases and 26 oral cancer cases from India. In addition, a group of five xeroderma pigmentosum (XP) patients were included in the study. We also analysed 26 different tumour cell lines. Blood samples were collected from both cases and controls with the exception of melanoma cases. DNA was isolated from blood samples using a phenol–chloroform extraction method (Kumar and Hemminki, 1996
). In the case of melanomas, DNA was isolated from paraffin embedded tumour tissues using a proteinase K digestion method (Kumar et al., 1998).
Genotyping
Exon 1 of the p53R2 gene along with flanking non-coding sequences was amplified as a 249 bp fragment using the forward primer 5'-GGA CAG GCG AGA AAG CAG GAC and the reverse primer 5'-TGA GGG GGA AGA CGC AAC AG. Each 10 µl reaction contained 50 mM KCl, 1.0 mM MgCl2, 0.11 mM each dNTP, 1 µCi [
-32P]dCTP, 0.3 µM each primer and 0.5 U Platinum Taq polymerase (Life Technologies, Gaithersburg, MD). Temperature conditions were 95°C for 45 s, 66°C for 45 s, 72°C for 45 s for 3 cycles followed by 95°C for 20 s, 65°C for 20 s, 72°C for 20 s for 32 cycles and a final extension at 72°C for 7 min. The 8 bp insertion polymorphism was initially detected by PCR–SSCP as previously described (Smeds et al., 2001) and confirmed by direct sequencing. Genotyping of the different populations was carried out on 6% denaturing polyacrylamide gels, where polymorphic cases carrying the 8 bp insertion could be separated from the wild-type cases, based on fragment size.
Sequence analysis
The detected polymorphism was confirmed by sequencing. DNA was extracted from the shifted band on the gel, amplified by PCR and purified using Sephadex micro-spin columns (Amersham Pharmacia Biotech, Uppsala, Sweden). The purified PCR product was subjected to 26 cycles of sequencing reactions using reverse and forward primers separately. The precipitated sequencing reaction products was electrophoresed on a denaturing polyacrylamide gel in an automated sequencer (ABI 377; Applied Biosystems, Foster City, CA) and analysed using Edit View 1.0.1 software (Applied Biosystems, Foster City, CA). The sequencing data were analysed using Align software in DNA Star package (DNASTAR Inc, Madison, WI).
Statistical analysis
Statistical significance of differences between frequency of polymorphism, in the different populations, was determined using the 2-test or Fisher's exact test. In addition, odds ratios (OR) were determined for the frequency of polymorphism between the different populations (dos Santos Silva, 1999). 2-test was also utilized in comparisons of genotype frequencies for the different ethnic groups with expected Hardy–Weinberg proportions.
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Results |
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We have detected a novel 8 bp insertion polymorphism in the 5'-UTR of the recently cloned gene p53R2 located on chromosome 8q23.1. Insertion of the 8 bp sequence 5'-GCGGACCA-3' created an additional repeat where two direct tandem repeats pre-existed between nucleotides –95 and –78 (relative to ATG translation initiation codon) (Figure 1
). The previously reported C
A polymorphism (Smeds et al., 2001) at position –88 is located within the first of the direct tandem repeats and causes the loss of exactness in these direct repeats. In none of the individuals analysed for both the 8 bp insertion and –88 CA base change were the two polymorphisms found to co-segregate.
The polymorphic frequencies in different cancer cases from Nordic population were compared with healthy controls from the same population as shown in Table I. The frequency of the allelic variant was lower in melanoma cases (0.02) than in controls (0.07) but the difference was not statistically significant (2 = 3.28; P = 0.07; OR = 0.32; 95% CI 0.07–1.26). Moreover, the number of melanoma cases with polymorphism (n = 3) was small. Two out of five XP patients were heterozygotes for the variant allele. We tested 26 different tumour cell lines for polymorphism and this insertion polymorphism was found in four cell lines. One cell line was found to have an insertion consisting of two 8 bp repeats, thus resulting in four tandem repeats (Figure 1E).
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The various European populations showed polymorphic frequencies similar to each other, ranging from 0.06 to 0.09. Among Asian populations the frequencies were 0.03 in both Japanese and Chinese populations, while none of the Indian individuals were polymorphic. The collective frequency of polymorphism for the Caucasian population, 0.07, was significantly higher than the frequency (0.02) that was observed for all Asian individuals collectively (Table II
; 2 = 9.19; P = 0.002; OR = 3.13; 95% CI 1.39–7.43). In Caucasian populations the genotype frequencies did not show significant deviations from the Hardy–Weinberg equilibrium; however, in the Asian population, the number of homozygotes with the 8 bp insertion was more than expected (Table II). Compilation of all 843 individuals included in the study resulted in the average polymorphic frequency of 0.06.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Our study describes a novel 8 bp insertion polymorphism, adjacent to two identical 8 bp tandem repeats in the 5'-UTR of the recently cloned p53R2 gene. We had previously reported a –88 C
A 5'-UTR polymorphism in this gene, which is located within the first copy of the two 8 bp tandem repeats (Smeds et al., 2001). Polymorphisms located in promotor regions may cause deviation in regulation of transcription. The effect of the polymorphisms detected in the 5'-UTR of the p53R2 gene remains to be determined, as the promoter of the gene has not been studied for location of transcription binding sites. Altered ribonucleotide reductase levels have been demonstrated to be involved in tumorigenicity and metastasis (Fan et al., 1996; Chen et al., 2000) and since the small subunit of the enzyme is critical for activity of the enzyme, alterations in p53R2 activity could have a significant impact on carcinogenesis. Inhibition of dNTP synthesis blocks replication of cancer cells while active ribonucleotide reductase conversely may increase dNTP production and stimulate cell division (Abid et al., 1999). Though in the present study we detected a lower frequency of polymorphism in melanoma patients than in healthy individuals, the difference was not significant. The effect of insertion of the 8 bp direct repeat on the expression of the gene in melanomas could not be ascertained due to the inability to isolate RNA from poor quality paraffin-embedded tissues. The number of XP cases analysed in the study was limited to five, incidentally, out of which two were found to be heterozygotes. Further investigation of the role of p53R2 in the DNA repair deficiency syndrome might be interesting. In general the genotype frequency of the 8 bp insertion in the 5'-UTR of p53R2 corresponded to Hardy–Weinberg equilibrium except for Chinese and Japanese healthy controls. However, in order to verify a true deviation from Hardy–Weinberg equilibrium an extended study with a larger number of individuals from these populations than in the present study would be required. Since there were no significant differences in the frequency of the 8 bp insertion between cancer cases and healthy controls, we presumed therefore that the allelic frequencies observed in both represent the population frequency. The only significant difference observed in the allelic frequencies was between populations. The frequency of the 8 bp insertion in p53R2 was significantly higher in Caucasians than in the Asian population.
Direct tandem repeats constitute a measure of homologous recombination events such as strand exchange and non-conservative processes including single strand annealing and replication slippage (Lambert et al., 1999). Homologous recombination is involved in cell cycle control and DNA repair, but can also contribute to genome instability by creating genomic changes such as duplications and deletions. About 10% of mutations in the p53 gene have been reported to occur by insertions or deletions involving repeat sequences (Jego et al., 1993). Other genes in which duplicating insertions and deletions at sites of direct repeats have been described are RB1, APC, HPRT and prohibitin (Canning and Dryja, 1989; Nishisho et al., 1991; Sato et al., 1992; Osterholm et al., 1996). Thus the 8 bp tandem repeats located in the 5'-UTR of the p53R2 gene might constitute another unstable locus in the human genome, which is supported by detection of two additional tandem repeat insertions in a cancer cell line.
In summary, we report the identification of a novel 8 bp insertion polymorphism at the site of two pre-existing direct tandem repeats in the 5'-UTR of p53R2. Although we did not find any association between this polymorphism and the various cancers studied, the role of this insertion polymorphism on transcription or translation of the p53R2 gene remains to be determined.
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Acknowledgments |
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This study was supported by a grant from Swedish Cancer Society, Stockholm.
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Notes |
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1 To whom correspondence should be addressed. Email: johanna.smeds{at}cnt.ki.se
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References |
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Received on June 8, 2001; accepted on July 27, 2001.
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