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Epidemiology and Prevention |
1 Institute of Biomedical Sciences and 2 Life Science Library, Academia Sinica, Taipei; 3 Department of Surgery, Changhua Christian Hospital, Changhua; and 4 Department of Surgery, Tri-Service General Hospital, Taipei, Taiwan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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12 years between menarche and first full-term pregnancy) and a higher number of high-risk genotypes of the NHEJ genes was only seen in women with at least one variant BRCA1 allele (i.e., the Glu/Gly or Gly/Gly forms of BRCA1 Glu1038Gly); and (b) a phenotype-based study measuring in vitro and in vivo NHEJ capacity showed that the precise end-joining capacity was different in breast cancer cell lines with different BRCA1 statuses being higher in BRCA1-expressing MCF-7 cells than in HCC1937 cells (defective BRCA1 expression). Furthermore, this end-joining capacity was decreased in MCF-7 cells in which BRCA1 expression was blocked using small interfering RNA and increased in HCC1937 transfected with full-length BRCA1. Because BRCA1 is a well-documented breast cancer susceptibility gene, this association between NHEJ and BRCA1 not only suggests a role of BRCA1 in NHEJ but also provides essential support for the tumorigenic contribution of NHEJ in breast cancer development. |
INTRODUCTION |
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MATERIALS AND METHODS |
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20%) of all breast cancer cases diagnosed during the study period in these regions.
To avoid any differential recall bias of previous disease history, we deliberately randomly selected the controls from the health examination clinics of the same hospitals during the same study period. These controls accounted for 80% of all women attending the clinics, and no significant differences were found in socioeconomic status between those included and those not included. The control subjects underwent a 1-day comprehensive health examination and showed no evidence of breast cancer, suspicious precancerous lesions of the breast, or other cancers.
Three experienced research nurses were assigned to administer a structured questionnaire to both cases and controls to collect all relevant information regarding risk factors of breast cancer. A 10-ml sample of peripheral blood, collected in acetate-citrate dextrose, was obtained from each breast cancer patient before operation and from each control subject. Buffy coat cells were immediately prepared and stored at –80°C until genomic DNA was prepared by conventional phenol/chloroform extraction, followed by ethanol precipitation, and stored at –20°C.
Three SNPs in the BRCA1 gene, Pro871Leu, Glu1038Gly, and Ser1613Gly, were selected for study, as previous epidemiological studies had suggested a putative association between these specific SNPs and increased cancer risk (15) . The SNPs in the five NHEJ genes (Ku70, Ku80, DNA-PKcs, XRCC4, and Ligase IV) used have been described previously (11) . All SNPs were genotyped using a MassARRAY system (SEQUENOM, Inc., San Diego, CA) based on the primer extension protocol (16) . The PCR primers and extension primers for these two SNPs were designed using Spectro-Designer software (SEQUENOM, Inc.).
The association between putative high-risk genotypes of these five NHEJ genes and breast cancer risk was evaluated with simultaneous consideration of established risk factors for breast cancer in a multivariate logistic regression model (17) . Of particular interest was the possible modification by BRCA1 of the risk associated with the number of high-risk NHEJ genotypes, which was examined using a stratified method (18 , 19) and evaluated by calculating the risk (adjusted odds ratio and 95% confidence interval) of breast cancer in relation to harboring a higher number of high-risk NHEJ genotypes within women of different BRCA1 genotypic status. We also used the same stratified method to estimate the joint effect of NHEJ genes and reproductive risk factors because reproductive risk factors reflect a longer period of or higher susceptibility to exposure to estrogen, which is known to initiate breast cancer development by causing DNA DSBs (20 , 21) . Thus, if BRCA1 were involved in NHEJ, the joint effect would not be the same in women harboring different BRCA1 genotypes.
In Vivo and in Vitro Functional Assays of End-Joining Capacity.
To examine whether BRCA1 was involved in NHEJ, we measured end-joining capacity in vivo and in vitro in human breast cancer cells with either normal BRCA1 expression (MCF-7) or defective BRCA1 expression (HCC1937). Furthermore, to determine whether the association between BRCA1 and NHEJ was causal, in vitro and in vivo end-joining capacity was also measured in MCF-7 cells in which BRCA1 expression was blocked using small interfering RNA (siRNA) and in HCC1937 cells transiently transfected with BRCA1.
Cell Culture and Reagents.
The human breast cancer cell lines, MCF-7 and HCC1937, were purchased from the American Type Culture Collection (Manassas, VA) and grown in DMEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 mg/ml streptomycin, and 100 units/ml penicillin. The cell lines were cultured at a constant temperature of 37°C in a 5% carbon dioxide-humidified atmosphere.
Transfection with siRNA.
The siRNA duplexes consisted of 21 bp, including a two-deoxynucleotide overhang, the coding strand being GGAACCUGUCTCCACAAAGdTdT. Cells (MCF-7) were transfected with siRNA using the Oligofectamine protocol (Invitrogen-Life Technologies, Inc., La Jolla, CA). In brief, 24 h before transfection, the cells were seeded, without antibiotics, at 1 x 106 cells/60-mm dish, corresponding to 40–50% confluency at the time of siRNA transfection. The cells were then incubated for 4 h at 37°C with various concentrations of siRNA plus Oligofectamine in Opti-MEM I or with Opti-MEM I alone, then fresh culture medium containing 30% fetal bovine serum was added in the ratio of 1 volume/3 volumes of the transfection medium. The cells were harvested 24 h after the beginning of the transfection period for BRCA1 protein detection (by Western blotting) or in vitro NHEJ assay (13)
or additionally transfected with pGL2 for the in vivo end-joining assay (Ref. 14
; see below).
Full-Length BRCA1 Plasmid Transfection.
Parental HCC1937 cells were transfected with a pcDNA 3.1 plasmid containing the full-length BRCA1 gene. For transient transfectants, cells were transfected with full-length BRCA1 plasmid using the Lipofectamine 2000 protocol (Invitrogen). HCC1937 cells at 90–95% confluency were incubated for 4 h at 37°C with full-length BRCA1 plasmid plus Lipofectamine in Opti-MEM I or with Opti-MEM I alone, then fresh culture medium containing 30% fetal bovine serum was added in the ratio of 1 volume/3 volumes of the transfection medium. The cells were then processed as in the siRNA experiment above.
Western Blot Analysis of BRCA1 and Other DNA DSB Repair Proteins.
Protein extracts were prepared, and equal amounts of protein (50 µg) were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Hybond ECL Nitrocellulose Membrane; Amersham Pharmacia Biotech, Piscataway, NJ). After addition of the blocking mixture, the membrane was incubated for 2 h at room temperature with primary antibody and washed three times with PBS, then bound antibody was detected using horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Antibodies against RAD51, Ku70, BRCA1, XRCC4, and DNA Ligase IV were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA), whereas the anti-NBS1 antibody was purchased from Oncogene (Oncogene Research Products, La Jolla, CA). The signal was detected using enhanced chemiluminescence (Santa Cruz Biotechnology) on Kodak BioMax films (Sigma Chemical Co., St. Louis, MO).
In Vitro NHEJ Assay.
The cells were harvested and washed once in DMEM containing 10% FCS, three times in ice-cold PBS, and once in hypotonic lysis buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 5 mM DTT], then resuspended in 2 volumes of hypotonic buffer, incubated for 20 min at 0°C, and lysed by homogenization, then protease inhibitors (0.17 mg/ml phenylmethylsulfonyl fluoride, 0.01 trypsin inhibitor units/ml aprotinin, 1 µg/ml pepstatin, 1 µg/ml chymostatin, and 1 µg/ml leupeptin) were added. After 20 min on ice, 0.5 volumes of high salt buffer [50 mM Tris-HCl (pH 7.5), 1 M KCl, 2 mM EDTA, and 2 mM DTT] were added and the extract centrifuged for 3 h. The supernatant was then dialyzed for 3 h against dialysis buffer [20 mM Tris-HCl (pH 8.0), 0.1 M KOAc, 20% (v/v) glycerol, 0.5 mM EDTA, and 1 mM DTT], fast-frozen, and stored at –70°C. The pBSK(+) duplex plasmid DNA (2.96 kb; Stratagene, La Jolla, CA) was linearized with EcoRI, dephosphorylated using calf intestinal phosphatase, and 5'-32P end-labeled using polynucleotide kinase. Cell-free extracts (50–100 µg of protein) were incubated for 5 min at 37°C before being added to the reaction mix of 50 mM triethanolamine-HCl (pH 7.5), 0.5 mM Mg(OAc)2, 60 mM KOAc, 2 mM ATP, 1 mM DTT, and 100 µg/ml BSA containing 50 fmol of labeled linearized DNA. After incubation at 37°C for 1 h, the 32P-labeled DNA products were deproteinized for 20 min at 37°C using 500 µg/ml proteinase K and 1% SDS and analyzed by electrophoresis through 0.7% agarose gels.
The monomer and multimers bands (substrate and products) were detected on Kodak BioMax films (Sigma Chemical Co.). The data were quantitated by densitometry and, after normalization for loading and transfer, expressed as end-joining efficiency calculated as: intensity of end-joining products/total substrate x 100%.
In Vivo End-Joining Assay.
Plasmid pGL2 (Promega, Madison, WI) was completely linearized using either HindIII or EcoRI, as confirmed by agarose gel electrophoresis. The linearized DNA was subjected to phenol/chloroform extraction, ethanol-precipitated, dissolved in sterilized water, and used to transfect cells using Lipofectamine 2000 following the procedures described by the supplier (Invitrogen). The transfectants were harvested 48 h after transfection and assayed for luciferase activity as described previously (22)
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 test) in both cases and controls, and the frequency distribution based on haplotypes was similar to that based on individual SNPs. We therefore chose one SNP, Glu1038Gly, which showed the most significant P, to represent the allelic status of BRCA1 and investigated the potential importance of BRCA1 in conjunction with the high-risk genotypes of the five NHEJ genes by the stratified method. The heterozygous and homozygous variant genotypes (i.e., Glu/Gly, Gly/Gly) of BRCA1 were then grouped together and compared with the homozygous wild-type genotype (Glu/Glu); due to the small percentage of subjects with the homozygous variant genotype, this grouping provided increased statistical power. In agreement with our previous study using a smaller sample size (11)
, when the five NHEJ genes were considered as a whole, one additional putative high-risk NHEJ genotype was associated with a 1.17-fold (95% confidence interval, 1.01–1.34) increase in risk. However, interestingly, after stratification on the basis of BRCA1 genotypes, the risk (adjusted odds ratio) associated with harboring one additional high-risk NHEJ genotype was not the same in women with different BRCA1 genotypes, which was more significant in those with at least one variant allele of BRCA1 (Table 1)
. Furthermore, because estrogen is suggested to initiate breast tumorigenesis by causing DNA DSBs (20
, 21)
, the significant modification by BRCA1 of breast cancer risk associated with NHEJ genotypic polymorphism prompted us to examine whether a higher risk of breast cancer due to a longer period of estrogen exposure [reflected by a longer interval of 12 years between the ages of menarche and first full-term pregnancy] and defective DSB repair (indicated by harboring a higher number of putative high-risk genotype of NHEJ genes) was affected by BRCA1. The results, shown in Table 2
, demonstrated that a significant increase in cancer risk associated with the joint effect of having reproductive risk factors and a higher number of high-risk genotypes of the NHEJ genes was only seen in women with at least one variant BRCA1 allele.
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. In addition, measurement of the expression of other DSB repair proteins, including NHEJ pathway proteins (Ku70, Ligase IV, and XRCC4), DSB trimming proteins (NBS1), and a HR pathway protein (RAD51), showed no significant differences, except that XRCC4 expression was slightly reduced in BRCA1-restored HCC1937 cells (Fig. 1)
. These results emphasize the fact that any changes in in vivo and in vitro end-joining activities observed in the subsequent analysis were more likely to be caused by BRCA1 than by the other DSB repair proteins, levels of which were not significantly affected by BRCA1 modification.
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. To examine the role of BRCA1, we directly compared the NHEJ capacity of whole cell extracts of BRCA1-defective HCC1937 cells with that of MCF-7 and found that the HCC1937 cells showed a significant lower capacity, 80 µg of HCC1937 extract having only one-third to one-fourth of the capacity of the MCF-7 extract (Fig. 2A)
. The validity of the assay was reconfirmed by the dose-response relationship between the amount of whole cell extract from HCC1937 and the in vitro end-joining capacity (Fig. 2A)
.
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. In contrast, when the plasmid containing the full-length BRCA1 gene was transfected into HCC1937, transfection with 8 µg of plasmid caused an increase in end-joining capacity, whereas transfection with 16 µg increased the activity in HCC1937 cells to 70% of that in MCF-7 cells (Fig. 2B)
. The increasing and decreasing trends observed in these assays were all statistically significant (P < 0.05, examined by linear regression). Simultaneously performed immunoblots showed, as in Fig. 1
, that the levels of BRCA1 decreased or increased, respectively, in a dose-response manner using the loss-of-function or gain-of-function strategy (data not shown).
BRCA1 Affects in Vivo NHEJ Capacity.
We then examined whether the role of BRCA1 in affecting end-joining capacity was important in vivo. To detect end-joining capacity in vivo, a linearized pGL2 plasmid containing the endonuclease-digested luciferase reporter gene was transfected into breast cancer cell lines, the rationale being that the luciferase gene would only be expressed after the plasmid was re-end-joined as the circular form, and the relative end-joining efficiency could be calculated by comparing the luciferase activity in cells transfected with endonuclease-digested DNA with that in cells containing the uncut plasmid. The end-joining capacity detected using pGL2 digested with HindIII reflects overall end-joining (14)
because this enzyme cleaves at the linker region between the promoter and the coding sequences, and any end-joining activity, even that resulting in small deletions or insertions, would not affect luciferase expression. However, because the EcoRI site is in the luciferase sequence and only precise end-joining would restore the original luciferase, the relative luciferase activity using this enzyme reflects precise end-joining capacity (14)
.
After the same strategy used to define the role of BRCA1 in in vitro end-joining, we examined end-joining capacities in MCF-7 and HCC1937 breast cancer cell lines and found that, although their in vivo overall end-joining capacities were very similar, MCF-7 cells had significantly higher in vivo precise end-joining capacity than HCC1937 cells (Fig. 3A)
. Consistent with the results for the in vitro assay, the BRCA1-deficient HCC1937 cells re-gained precise end-joining capacity on transfection with the full-length BRCA1 plasmid, and the precise end-joining capacity of the BRCA1-expressing MCF-7 cells was eliminated by BRCA1 siRNA inhibition, both in a dose-response manner (Fig. 3B)
. The increasing and decreasing trends observed in these assays were all statistically significant (P < 0.05, examined by linear regression). Neither manipulation had any effect on in vivo overall end-joining activity (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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70%) of the genome in mammalian cells consists of extragenic sequences that do not code for protein amino acids (25)
, the trade-off between the use of NHEJ to fix DSB, with the relatively minor cost of imprecise repair, and efficient DSB repair to avoid lethality caused by unrepaired damage becomes acceptable. However, it would be mechanistically reasonable if there were additional factors that could improve the fidelity of NHEJ, and interestingly, BRCA1, one of the key members of HR, has been suggested to possess this function in studies on mouse embryonic fibroblasts and in lymphoblastoid cell lines established from women carrying BRCA1 mutation (13
, 26)
. The present study is the first to confirm this accessory role of BRCA1 in NHEJ in breast cancer cell lines, both in vitro and in vivo, and, given that BRCA1 is a breast cancer susceptibility gene, the results are more biological relevance than those in the previous studies on embryonic fibroblasts and cell lines. More importantly, we have also provided evidence that the involvement of BRCA1 in NHEJ is of tumorigenic significance and that the breast cancer risk associated with harboring a higher number of high-risk genotypes of NHEJ genes is significantly modified by BRCA1 polymorphism. Support for the role of BRCA1 in DNA DSB repair is mainly based on the fact that BRCA1 interacts directly or indirectly with the other proteins that play important roles in DSB repair (8 , 27) . Because it colocalizes with RAD51, an essential protein in regulating recombination and coforms discrete nuclear foci with RAD51 in intranuclear structures after treatment with DSB-causing agents (28) , BRCA1 has been suggested to be involved in controlling HR in DSB repair (8 , 23) . In addition, when cells are subjected to ionizing radiation, wild-type BRCA1 forms a complex with RAD50-MRE11-p95/NBS1 (MRN), both in vitro and in vivo, and the formation of the MRN complex is dramatically reduced in BRCA1-deficient breast cancer cells and restored by transfection with wild-type BRCA1 (29) . This suggests that BRCA1 is important for cellular responses to DNA damage mediated by the MRN complex. However, whether and how MRN is involved in DSB repair by NHEJ is still not resolved (30) , leading to speculation that BRCA1 might not play a significant role in the NHEJ pathway. Different studies based on in vivo or in vitro assays of NHEJ capacity in cell lines of different tissue origins have yielded inconsistent results, which may be due to differences in the host cells used, the type of end-joining capacity detected, and the nature of the Brca1 mutation in the mouse cells used (7 , 23 , 31) . To resolve these inconsistencies, we propose the possibility that more than one NHEJ mechanism exists and that BRCA1 is only required in some of these. As we demonstrated in the present study, the dependence of NHEJ on BRCA1 is condition-specific because the in vivo studies showed that BRCA1 was only required for precise end-joining.
The sample size of this case-control study is relatively large but, as many other association studies, might be still subject to possible underpower to detect the associations. However, because, in this study, the five NHEJ pathway genes were considered as a single unit (rather than as separate genes) and we did not separately examine which NHEJ gene was more important or which NHEJ gene interacted with BRCA1, we actually only examined two genetic risk factors, i.e., one gene (BRCA1) and one pathway (NHEJ). Because of this, sample size and underpowering might not be a serious concern, if at all.
The genotypic polymorphisms at BRCA1 and the NHEJ genes examined in the present study have been individually shown to be associated with breast cancer risk (11 , 15) . Although some polymorphisms are exonic and thus have the potential to alter the activity of enzymes, there is currently no evidence for genotype-phenotype associations, and thus, how the risk is affected by these SNPs remains unclear. It is possible that our epidemiological findings may be explained by alternative mechanisms such as (a) the polymorphisms may be in linkage disequilibrium with an exon change in the same gene that affects protein function, (b) intronic changes in gene sequences contain regulatory sequences such as enhancers, which affect the level of expression through transcriptional regulation, (c) the SNPs in these genes may be linked with alterations in other adjacent unidentified genes, increasing breast cancer risk. To exclude the third possibility, we attempted to use more than one SNP in these genes to assign the haplotypes and to examine haplotype effects on cancer risk, but the information generated by haplotype analysis was limited due to strong linkage disequilibrium between the SNPs in the same gene. Despite this, the strength of the present study is that it is based on the candidate gene approach, and, given the fact that all breast cancer susceptibility genes (including ATM, p53, PTEN, BRCA1, and BRCA2) are involved in DNA DSB checkpoint/repair pathway (6) , the study of an association between breast cancer development and an interaction between DSB repair genes (such as BRCA1 and NHEJ genes) is mechanistically and biologically reasonable. Although cell line studies and SNP-based epidemiological observations have provided evidence for this interaction between BRCA1 and NHEJ, animal models using knockout mice and familial breast cancer syndromes have not reported any such relationship. We appreciate that this difference is important and consider that it may shed light on the nature of the critical mechanisms by which the interaction between BRCA1 and NHEJ displays its tumorigenic contribution. We have proposed a hide-then-hit model (11) , suggesting that, for these high-penetrant genes, only subtle defects arising from low-penetrance (risk) alleles (e.g., polymorphic variant detected in the present study) would have the chance to escape cell-cycle surveillance without triggering checkpoint-induced lethality, thus leading to the accumulation of the essential genetic changes required for tumor formation. The epidemiological observations reported in this and our recent study (11) confirm such a hypothesis. It is also reasonable to speculate that mechanisms operating at the transcriptional level may be responsible for a similar low-penetrance effect in cancer development because they can switch genes on as well as off, thus modifying the level of expression of normal genes, instead of totally and permanently abolishing expression (32) . Accordingly, as compared with the previous study using a BRCA1-knockout mouse cell line bearing a p53 mutation to avoid cell death (13) , the demonstration in the present study, using both gain-of-expression (using transfection to increase BRCA1 expression) or loss-of-expression (using siRNA to knockdown BRCA1 expression), that the interaction between BRCA1 and NHEJ affects DSB repair capacity is more relevant to tumorigenesis. However, because their effects are low penetrant, these alleles would predispose carriers to a higher risk of developing cancer but not necessarily cause cancer. The probability of manifesting the tumorigenic phenotype depends on the interaction between these alleles and the environment. Recently, the role of reproductive hormones in breast tumorigenesis has been greatly expanded to suggest that estrogen might be a complete carcinogen directly causing genetic alteration and tumor initiation, and breast cancer can be initiated by estrogen via exposure to estrogen metabolites, including catechol estrogen and catechols estrogen quinones (20 , 21) . These metabolites cause DNA DSBs formation, which may impose a selective microenvironment in the breast epitheliums, explaining why the increased cancer risk associated with the interaction between BRCA1 and NHEJ varies in women with different profiles of estrogen-related risk factors.
The genetic evidence from familial cancer syndromes provides solid support for a causal relationship between mutated genes and the incidence of cancer, but not all cancer-associated genes can meet this requirement of linking germ-line defects to cancer development. However, as our understanding of tumorigenesis is extended from single-gene mechanisms to multigenic or to etiological pathway-wide networks, the consideration of whether there is a causal link between a putative cancer-associated gene and tumor development might be extended to whole tumorigenic networks. The most recent example is the discovery of the BRCA2-interacting protein, EMSY, which binds to BRCA2 and turns off expression of BRCA2, and the demonstration that amplification of EMSY is frequently seen in sporadic breast cancer and is associated with worse survival (33) . This finding has therefore been considered to provide the missing link between BRCA2 and sporadic breast cancer development. Similarly, the finding that the interaction of NHEJ with BRCA1 is associated with both increased risk of breast cancer and precise end-joining capacity not only suggests a role of BRCA1 in NHEJ, but it also provides essential support for tumorigenic contribution of NHEJ during breast cancer formation.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Chen-Yang Shen, Institute of Biomedical Sciences, Academia Sinica, Taipei, 11529 Taiwan. Phone: 886-2-2789-9036; Fax: 886-2-2782-3047; E-mail: bmcys{at}ccvax.sinica.edu.tw
5 Internet address: http://linkage.rockefeller.edu/soft. 
Received 2/ 6/04. Revised 4/16/04. Accepted 5/17/04.
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