© 2005 Foundation for Promotion of Cancer Research
Cancer Genetics Report |
Genetic Alteration of Poly(ADP-ribose) Polymerase-1 in Human Germ Cell Tumors
1 Biochemistry Division, National Cancer Center Research Institute, Tokyo, 2 The 1st Department of Obstetrics and Gynecology, Toho University School of Medicine, Tokyo, 3 The Department of Neurosurgery, Dokkyo University School of Medicine, Iruma-gun, Saitama and 4 The Department of Neurosurgery, Saitama Medical University, Shimotsuga-gun, Tochigi, Japan
For reprints and all correspondence: Dr Mitsuko Masutani, Biochemistry Division, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. E-mail: mmasutan{at}gan2.res.ncc.go.jp
Received October 8, 2004; accepted December 11, 2004
 |
Abstract |
|---|
 TOP Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Accumulated evidence suggests that poly(ADP-ribose) polymerase-1 (PARP-1) is involved in DNA repair, cell-death induction, differentiation and tumorigenesis. Parp-1 deficiency also induces trophoblast differentiation from mouse embryonic stem cells during teratocarcinoma-like tumor formation. To understand the relationship of PARP-1 dysfunction and development of germ cell tumors, we conducted a genetic analysis of the PARP-1 gene in human germ cell tumors. Sixteen surgical specimens of germ cell tumors that developed in the brain and testes were used. Two known single nucleotide polymorphisms (SNPs) (Val762Ala and Lys940Arg), which are listed in the SNP database of the NCBI (National Center for Biotechnology Information), were detected. In both cases, cSNPs encoded amino acids located within peptide stretches in the catalytic domain, which are highly conserved among various animal species. Furthermore, another novel sequence alteration, a base change of ATG to ACG, was identified in a tumor specimen, which would result in the amino acid substitution, Met129Thr. This base change was observed in one allele of both tumor and normal tissues, suggesting that it is either a rare SNP or a germline mutation of the PARP-1 gene. Notably, the amino acid Met129 is located within the second zinc finger domain, which is essential for DNA binding and is conserved among animal species. One SNP in intron 2 and one in the upstream 5'-UTR (untranslated region) were also detected.
Key Words: brain • germ cell tumor • mutations • poly(ADP-ribose) polymerase-1 • SNP
|
INTRODUCTION |
|---|
|
TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Poly(ADP-ribose) polymerase (PARP-1) catalyzes polyADP-ribosylation of various proteins (1), including histones, PARP-1 itself, X-ray repair cross-complementing factor-1 (XRCC-1) (2), NF-
B (3) and p53 (4), using nicotinamide adenine dinucleotide (NAD) as a substrate. PARP-1 is present in nuclei and centrosomes (5) and is composed of three domains, namely: DNA-binding, automodification and NAD-binding domains. PARP-1 is activated by DNA strand-breaks and is involved in DNA repair (6–8) and also in the maintenance of genomic stability (9,10). PARP-1 takes part in cell-death induction through depletion of cellular NAD levels (11) and by activating apoptosis-inducing factor 1 (12). Parp-1–/– mice show a higher susceptibility to carcinogenesis induced by alkylating agents (13,14). PARP-1 also participates in the transcriptional regulation of some genes (15–17) and in cellular differentiation (18–20). Parp-1–/– mouse embryonic stem (ES) cells show preferential induction of the trophoblast lineage (17), leading to trophoblast giant cells (TGCs) after subcutaneous injection into nude mice (20). The biochemical properties of TGCs resemble syncytiotrophoblastic giant cells (STGCs) of human germ cell tumors. It is thus suggested that PARP-1 deficiency may possibly trigger differentiation of STGCs within germ cell tumors during tumor formation. The appearance of STGCs in some trophoblastic or choriocarcinomatous human germ cell tumors is reported to be associated with high metastatic potential and poor prognosis (21). It is also interesting to note that teratocarcinoma cells undergo differentiation in vitro, at least in part, in the presence of the PARP inhibitor 3-aminobenzamide (19). Therefore, Parp-1 could be involved in the development of teratocarcinomas.
The human PARP-1 gene was previously mapped to chromosome 1q41–q42 (22) and is 43 kb in length and contains 23 exons (23). There have been few reports concerning the structural and functional analysis of the human PARP-1 gene in tumors (24,25). Prasad et al. (26) showed overexpression of the PARP-1 gene in human Ewing's sarcoma, and Menegazzi et al. (27) observed increased expression of the PARP-1 gene in high-grade lymphoma. Bieche et al. (28) observed that lower expression of the PARP-1 gene is associated with reduction in genomic stability in human breast cancer. We recently reported the reduced expression and structural alteration of the PARP-1 gene in some human tumor cell lines (29).
In the present study, we analyzed the PARP-1 gene in human germ cell tumors in order to clarify the relevance of PARP-1 deficiency in the development of human germ cell tumors.
|
MATERIALS AND METHODS |
|---|
|
TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Germ cell tumors were obtained from patients who were treated at the Saitama Medical School Hospital, Saitama, Japan, the Kanazawa University Hospital and National Cancer Center Hospital, Tokyo, Japan. Specimens of germ cell tumors of the brain were taken from 14 patients and corresponding non-cancerous tissue from 13 patients (Table 1).
|
Oligonucleotide PCR primer sets for the 23 exons of the PARP-1 gene were designed from intron sequences of the respective exons based on the complete human PARP-1 sequence (Table 2). Each exon of PARP-1 was amplified by PCR using 50–300 ng of genomic DNA with primer sets at a 1 µM concentration and with 100 mU of Ex Taq polymerase (Takara BIO Inc., Tokyo, Japan) or LA Taq polymerase (Takara BIO Inc.). The PCR amplification was performed using a thermal cycler (Perkin Elmer) for 35 cycles with 94°C for 1 min, annealing performed at the respective temperatures indicated in Table 2, for 1 min, and 72°C for 1 min, after an initial hot start at 94°C for 5 min. PCR products were purified using DNA Clean & Concentrator-TM5 (Zymo Reseach, USA), and then directly sequenced for both strands using a DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech, USA). The residual dye terminator was removed using an Autoseq G-50 kit (Amersham Pharmacia Biotech), and samples were analyzed on an ABI Prism 310 Auto Sequencer (Applied Biosystems). Sequence comparison was carried out against the sequence of the human PARP-1 gene (accession no. NT004559) and its cDNA (accession nos M18112 [GenBank] , M32721 [GenBank] , M17081 [GenBank] , J03473 [GenBank] , BC037545 [GenBank] and BC014206 [GenBank] ).
|
|
RESULTS |
|---|
|
TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Thirteen of 16 germ cell tumors were analyzed for all 23 exons. Sequence alterations and single nucleotide polymorphisms (SNPs) found in the PARP-1 gene are listed in Table 3. Eight SNPs were detected and two SNPs accompanied amino acid substitution, Val762Ala and Lys940Arg, respectively, as shown in Fig. 1 and Table 3. Three brain tumors (B5, B6 and B13) and their normal counterparts contained Val/Ala heterozygous alleles at amino acid position 762. Two other brain tumors (B10 and B11) and their normal counterparts only possessed an Ala allele. Two brain tumors (B8 and B13) and their normal counterparts only contained an Arg allele at amino acid position 940. These two SNPs, Val762Ala and Lys940Arg, are already in the list of the NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nls.nih.gov/) of SNPs. Interestingly, a brain tumor (B10) contained a novel sequence alteration, ATG
ACG, causing a Met129Thr amino acid substitution in one allele, in both the tumor and normal counterpart (Fig. 1). This sequence alteration has not been listed in the NCBI database of SNPs, suggesting that it is either a rare allele or a germline mutation. Four SNPs, which did not cause amino acid substitution, were also found at Asp81, Ala284, Lys352 and Phe638 (Table 3). A novel sequence alteration of G to C was also found in the upstream 5'-UTR (untranslated region) 17 bases upstream of the translation initiation site, immediately downstream of a putative ETS-1-binding site (base –26 to –22) (30) in one testicular tumor (T1). Although the normal counterpart tissue for T1 was not available, we observed the non-heterozygous C allele also in normal colon tissue (data not shown), which suggests that this sequence alteration is an SNP. In addition, an SNP in intron 2, which is located six bases upstream of exon 3, was found.
|
|
|
DISCUSSION |
|---|
|
TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
In this study, we report a novel sequence alteration that causes amino acid substitution, namely Met129Thr, in the PARP-1 gene. This sequence alteration has not been reported as an SNP and could be a germline mutation. Met129 is located at the second zinc finger motif and a Met129Thr substitution may possibly affect the DNA binding activity of PARP-1.
Two SNPs that accompanied amino acid substitutions, Val762Ala and Lys940Arg, were found. Both amino acid residues are located in the catalytic domain of PARP-1 and are highly conserved among species. The incidence of the non-heterozygous GCG allele, which causes a Val762Ala amino acid substitution, was higher (normal, 15.3%; tumor, 12.5%) than the reported value of 5.9% in the SNP 500 Cancer Database of the NIH Cancer Genomic Anatomy Project (http://snp500cancer.nci.nih.gov). The incidence of the non-heterozygous AGG allele, which causes amino acid substitution Lys940Arg, was also higher than expected (normal, 15.4%; tumor, 12.5%) and the heterozygous allele was not detected in the 16 samples analyzed. This may well be caused by the small sample size. However, there is an alternative possibility that non-heterozygous alleles may promote development of germ cell tumors. It should be further investigated whether the Ala/Ala allele at amino acid residue 762 and the Arg/Arg allele at amino acid residue 940 could contribute to the development of germ cell tumors.
Recently, Lockett et al. (31) showed that the Ala/Ala genotype of the Val762Ala SNP in the PARP-1 gene is associated with an increased risk for prostate cancer in Caucasian subjects. They reported decreased PARP-1 activity of Ala/Ala compared to the Val/Val genotype. Detailed analysis of genetic effects of Val762Ala, Lys940Arg and Met129Thr on PARP-1 function may be helpful in understanding the significance of these SNPs in germ cell tumor development.
A promoter allelotype of PARP-1 is reported to relate to susceptibility to rheumatoid arthritis (32). In this study, we observed a novel sequence alteration of G to C at the 5'-UTR (see Table 3) in tumor sample T1. Although the normal counterpart tissue was not available for T1, this sequence alteration was likely to be an SNP because we found this non-heterozygous C allele in the normal colon tissue from an unrelated sample as well. This sequence alteration in the 5'-UTR, located 17 bases upstream from the first ATG, does not overlap with Kozak's consensus sequence (33), therefore the effect on translation is not currently understood well. The major transcription initiation site is present at 146 bases upstream of this sequence alteration. The sequence alteration by this SNP is located nine bases upstream of a putative binding site for transcription factor Ets-1 (30) and, in addition, this region is GC-rich. Therefore, if the alternative transcription starts from the nearby sequence, the transcription efficiency may be affected by the secondary structural changes. Further studies of the effect on the transcription and translation of the PARP-1 gene may facilitate our understanding of the role of the sequence alteration in the 5'-UTR.
We observed both a heterozygous and non-heterozygous SNP in intron 2 at the same frequency (3/16) in germ cell tumors. This SNP is located within close proximity to the splicing acceptor site for exon 3. Nakata et al. (34) reported that a relatively conserved stretch of (C/T)6NCAGG(C/T) at a splicing acceptor site is generally observed in the introns. The SNP at intron 2 in Table 3 located within this pyrimidine-rich stretch [TTTGATT(C/A)TCCAGG, where italic AG corresponds to the intronic sequence at the intron–exon boundary] and alteration of C to A may possibly affect the splicing efficiency at exon 3.
Germ cell tumors are known to be highly sensitive to chemotherapeutic agents, including cisplatin, and radiation therapies. Since PARP-1 is involved in DNA repair, dysfunction of PARP-1 may affect the outcome of cancer therapy besides its impact on cancer susceptibility, indicating that functional and genetic analysis of the PARP-1 gene is important not only for the elucidation of the mechanism of cancer development but also from a therapeutic point of view.
|
Acknowledgments |
|---|
This work was supported in part by a Grant-in-Aid for the Second Term Comprehensive 10-Year Strategy for Cancer Control and a Grant-in-Aid for Cancer Research from the Ministry of Health, Labour and Welfare of Japan. We are grateful to T. Otsubo for technical assistance on this study.
|
References |
|---|
|
TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
1 Rolli V, Rulf A, Augustin A, Schuls GE, Ménissier-de Murcia J, de Murcia G. Poly(ADP-ribose) polymerase: structure and function. In: de Murcia G, Shall S, editors. From DNA Damage and Stress Signaling to Cell Death. PolyADP-ribosylation Reactions. New York: Oxford University Press 2000;35–79.
2 Masson M, Niedergang C, Schreiber V, Muller S, Ménissier-de Murcia J, de Murcia G. XRCC1 is specifically associated with poly(ADP-ribose) polymerase and negatively regulates its activity following DNA damage. Mol Cell Biol 1998;18:3563–71.
3 Hassa PO, Hottiger MO. A role of poly(ADP-ribose) polymerase in NF-kappaB transcriptional activation. Biol Chem 1999;380:953–9.[CrossRef][Web of Science][Medline]
4 Vaziri H, West MD, Allsopp RC, et al. ATM-dependent telomere loss in aging human diploid fibroblasts and DNA damage lead to the post-translational activation of p53 protein involving poly(ADP-ribose) polymerase. EMBO J 1997;16:6018–33.[CrossRef][Web of Science][Medline]
5 Kanai M, Tong WM, Sugihara E, Wang ZQ, Fukasawa K, Miwa M. Involvement of poly(ADP-ribose) polymerase 1 and poly(ADP-ribosyl)ation in regulation of centrosome function. Mol Cell Biol 2003;23:2451–62.
6 Durkacz BW, Omidiji O, Gray DA, Shall S. (ADP-ribose)n participates in DNA excision repair. Nature 1980;283:593–6.[CrossRef][Medline]
7 Dantzer F, Schreiber V, Niedergang C, et al. Involvement of poly (ADP-ribose) polymerase in base excision repair. Biochimie 1999;81:69–75.[Medline]
8 Malagna M, Althaus FR. Poly(ADP-ribose) reactivates stalled DNA topoisomerase I and induces DNA strand break resealing. J Biol Chem 2004;279:5244–8.
9 Ménissier-de Murcia J, Niedergang C, Trucco C, et al. Requirement of poly(ADP-ribose) polymerase in recovery from DNA damage in mice and in cells. Proc Natl Acad Sci USA 1997;94:7303–7.
10 Wang ZQ, Stingl L, Morrison C, et al. PARP is important for genomic stability but dispensable in apoptosis. Genes Dev 1997;11:2347–58.
11 Berger NA. Poly(ADP-ribose) in the cellular response to DNA damage. Radiat Res 1985;101:4–15.[Web of Science][Medline]
12 Yu SW, Wang H, Poitras MF, et al. Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor. Science 2002;297:259–63.
13 Tsutsumi M, Masutani M, Nozaki T, et al. Increased susceptibility of poly(ADP-ribose) polymerase-1 knockout mice to nitrosamine carcinogenicity. Carcinogenesis 2001;22:1–3.
14 Nozaki T, Fujihara H, Watanabe M, et al. Parp-1 deficiency implicated in colon and liver tumorigenesis induced by azoxymethane. Cancer Sci 2003;94:497–500.[CrossRef][Medline]
15 Oei SL, Griesenbeck J, Schweiger M, Ziegler M. Regulation of RNA polymerase II-dependent transcription by poly(ADP-ribosyl)ation of transcription factors. J Biol Chem 1998;273:31644–7.
16 Simbulan-Rosenthal CM, Ly DH, Rosenthal DS, et al. Misregulation of gene expression in primary fibroblasts lacking poly(ADP-ribose) polymerase. Proc Natl Acad Sci USA 2000;97:11274–9.
17 Hemberger M, Nozaki T, Winterhager E, et al. Parp1-deficiency induces differentiation of ES cells into trophoblast derivatives. Dev Biol 2003;257:371–81.[CrossRef][Web of Science][Medline]
18 Terada M, Fujiki H, Marks PA, Sugimura T. Induction of erythroid differentiation of murine erythroleukemia cells by nicotinamide and related compounds. Proc Natl Acad Sci USA 1979;76:6411–4.
19 Ohashi Y, Ueda K, Hayaishi O, Ikai K, Niwa O. Induction of murine teratocarcinoma cell differentiation by suppression of poly(ADP-ribose) synthesis. Proc Natl Acad Sci USA 1984;81:7132–6.
20 Nozaki T, Masutani M, Watanabe M, et al. Syncytiotrophoblastic giant cells in teratocarcinoma-like tumors derived from Parp-disrupted mouse embryonic stem cells. Proc Natl Acad Sci USA 1999;96:13345–50.
21 von Hochstetter AR, Sigg C, Saremaslani P, Hedinger C. The significance of giant cells in human testicular seminomas. A clinico-pathological study. Virchows Arch A Pathol Anat Histopathol 1985; 407:309–22.[CrossRef][Web of Science][Medline]
22 Baumgartner M, Schneider R, Auer B, Herzog H, Schweiger M, Hirsch-Kauffmann M. Fluorescence in situ mapping of the human nuclear NAD+ ADP-ribosyltransferase gene (ADPRT) and two secondary sites to human chromosomal bands 1q42, 13q34, and 14q24. Cytogenet Cell Genet 1992;61:172–4.[Web of Science][Medline]
23 Auer B, Nagl U, Herzog H, Schneider R, Schweiger M. Human nuclear NAD+ ADP-ribosyltransferase(polymerizing): organization of the gene. DNA 1989;8:575–80.[Web of Science][Medline]
24 Masutani M, Nakagama H, Sugimura T. Poly(ADP-ribose) and carcinogenesis. Genes Chromosomes Cancer 2003;38:339–48.[CrossRef][Web of Science][Medline]
25 Masutani M, Gunji A, Tsutsumi M, et al. Role of poly-ADP-ribosylation in cancer development. In: Buerkle A, editor. Landes Bioscience Intelligence Unit Series, Poly(ADP-ribosyl)ation. Georgetown, Texas, USA Landes Bioscience/Eurekah Com 2004, in press.
26 Prasad SC, Thraves PJ, Bhatia KG, Smulson ME, Dritschilo A. Enhanced poly(adenosine diphosphate ribose) polymerase activity and gene expression in Ewing's sarcoma cells. Cancer Res 1990;50:38–43.
27 Menegazzi M, Scarpa A, Carcereri de Prati A, Menestrina F, Suzuki H. Correlation of poly(ADP-ribose)polymerase and p53 expression levels in high-grade lymphomas. Mol Carcinog 1999;25:256–61.[CrossRef][Web of Science][Medline]
28 Bièche I, de Murcia G, Lidereau R. Poly(ADP-ribose) polymerase gene expression status and genomic instability in human breast cancer. Clin Cancer Res 1996;2:1163–7.[Abstract]
29 Masutani M, Nozaki T, Sasaki H, et al. Aberration of poly(ADP-ribose) polymerase-1 gene in human tumor cell lines: its expression and structural alterations. Proc Japan Acad 2004;Series B 80:114–8.
30 Soldatenkov VA, Albor A, Patel BK, Dreszer R, Dritschilo A, Notario V. Regulation of the human poly(ADP-ribose) polymerase promoter by the ETS transcription factor. Oncogene 1999;18:3954–62.[CrossRef][Web of Science][Medline]
31 Lockett KL, Hall MC, Xu J, et al. The ADPRT V762A genetic variant contributes to prostate cancer susceptibility and deficient enzyme function. Cancer Res 2004;64:6344–8.
32 Pascual M, Lopez-Nevot MA, Caliz R, et al. A poly(ADP-ribose) polymerase haplotype spanning the promoter region confers susceptibility to rheumatoid arthritis. Arthritis Rheum 2003;48:638–41.[CrossRef][Web of Science][Medline]
33 Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res 1984;12:857–72.
34 Nakata K, Kanehisa M, DeLisi C. Prediction of splice junctions in mRNA sequences. Nucleic Acids Res 1985;13:5327–40.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  T. Ono, T. Kaneda, A. Muto, and T. Yoshida Positive Transcriptional Regulation of the Human {micro} Opioid Receptor Gene by Poly(ADP-ribose) Polymerase-1 and Increase of Its DNA Binding Affinity Based on Polymorphism of G-172 -> T J. Biol. Chem., July 24, 2009; 284(30): 20175 - 20183. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Li, X. Zhang, G. Huang, X. Miao, L. Guo, D. Lin, and S.-H. Lu Identification of a novel polymorphism Arg290Gln of esophageal cancer related gene 1 (ECRG1) and its related risk to esophageal squamous cell carcinoma Carcinogenesis, April 1, 2006; 27(4): 798 - 802. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||