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• W2093354896 abstract "Recent predictions of risks of developing a complex disease based on common genetic variations using breast cancer as a model suggest that the construction and use of genetic-risk profiles may improve the efficacy of population-based programs of intervention.1 However, the identification of genes, alleles or haplotypes predisposing to complex diseases including breast cancer has been difficult to accomplish, despite the existence of a significant proportion of familial cases of breast cancer that cannot be attributed to BRCA1 or BRCA2 mutations.2, 3 A major obstacle has been an insufficient statistical power of allelic association, linkage and population studies and a lack of functional assays for candidate allelic variants. Although common genetic variants are thought to confer a risk of breast cancer through multiplicative effects of a number of predisposing alleles,1 the significance of rare allelic polymorphisms has been particularly difficult to address because a prohibitive sample size would be required for allelic association studies. DNA helicases represent an important group of enzymes implicated in DNA repair, replication, recombination and transcription.4 Deficiencies of DNA helicases have been linked to cancer predisposition and premature aging as mutations in genes encoding the RecQ DNA helicases have been associated with cancer-predisposing syndromes, such as BLM in Bloom's syndrome, WRN in Werner's syndrome and RecQ4 in Rothmund-Thomson's syndrome.4, 5 In addition, mutations in 2 DNA helicase-encoding genes, XPB and XPD, have been linked to xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy,6 and polymorphisms in XPD have been associated with a risk of developing basal cell carcinomas and melanomas.7 A recently described nuclear DNA helicase BACH1,8 which has a high-sequence homology to a UV-induced protein SUVi at both protein and DNA levels,9 has been implicated in the repair of double-strand DNA breaks through its direct interaction with BRCA1. Two germline missense mutations were found in 65 breast cancer patients lacking BRCA1 and BRCA2 alterations,8 suggesting that BACH1 may be a breast cancer susceptibility gene. However, a follow-up study reported no mutations in the germline of 29 multiple-case breast cancer families with positive nonparametric linkage scores to short tandem repeats flanking the BACH1 locus at 17q and of 95 unrelated individuals with familial breast cancer who had no detectable mutation in BRCA1 and BRCA2.10 The latter study reported a 517C→T transition on 5 of 1,586 chromosomes in the Swedish population (allelic frequency 0.3%), leading to a substitution of arginine (R) 173 for cysteine (C) in a putative nuclear localization sequence (NLS) of BACH1.10 NLS is a short peptide sequence necessary and sufficient for nuclear localization of a protein,11 and single amino acid substitution involving critical residues in NLS has been shown to impair the nuclear localization.12 Deletion of arginine or lysine in the NLS of BLM resulted in an even distribution of the EGFP-BLM fusion protein in the cytoplasm and nucleus, whereas a substitution with a nonbasic amino acid reduced the nuclear localization of the fusion protein.13 The BACH1 517C→T polymorphism results in a replacement of charged R173 to uncharged polar C, thus potentially altering NLS, nuclear transport of BACH1 and may modify breast cancer susceptibility. To test whether BACH1 517C→T can inhibit the nuclear translocation of the enzyme, we generated BACH1-517T and BACH1-ΔNLS1 constructs using site-directed mutagenesis (SDM). The plasmid vector pcDNA3.1Myc-His/BACH1 containing full-length BACH1 cDNA was described previously8 and was kindly provided by Dr. S. Cantor (Harvard Medical School, Boston, MA). The BACH1 full-length cDNA was released by NotI/ApaI (Promega, Madison, WI) and subcloned into pGEM5Zf (Promega) to obtain suitable restriction sites for further cloning into the SDM vector. The recombinant molecule was digested with SphI/XbaI (Promega), and the resulting fragments sized 0.85 kb and 5.85 kb were recovered by gel purification. The 0.85 kb fragment was subcloned into pALTER-Ex1 (Promega) and the SDM was carried out using the Altered Sites® II in vitro Mutagenesis System (Promega). The SDM primer for creating BACH1-517T was: 5′-CAA AGC AAT GAC ATT TTC TAA TC; the SDM primer for eliminating NLS1 in BACH1-ΔNLS1 was: 5′-TTG TGT ACT TCT GTT CCA AAG CAA TTC TCT ACT TGA AAA TCA TCA T. The NLS1 in BACH1-ΔNLS1 was replaced with an asparagine (Fig. 1c). Following SDM, the 0.85 kb fragment was recovered and ligated with the 5.85 kb fragment to generate the mutant constructs. The wild-type BACH1, BACH1-517T and BACH1-ΔNLS1 were then released by digestion with SalI/ApaI (Promega) and subcloned into pEGFP-C2 (Clontech, Palo Alto, CA) for transfection experiments. The identities of all constructs were confirmed by direct sequencing as described previously14 prior to transfection. Confocal microscopy for cellular localization of BACH1. MCF-7 cells expressing wild-type BACH1-GFP (a), BACH1-517T-GFP (b), BACH1-ΔNLS1-GFP with asparagines (N) replacing NLS1 (c) and GFP (d). Samples were fixed 36 hr after transfection because no obvious differences in the GFP distribution were observed between the time points. Original magnification 630×. The corresponding constructs used for transfection experiments are shown in the right panel. For transient transfections, the MCF7 cell line was propagated in DMEM with 10% fetal calf serum and 100 nM estradiol, then plated on sterile cover slips in a 6-well plate. Five micrograms of the wild-type BACH1, BACH1-517T and BACH1-ΔNLS1 plasmid constructs were used for transfections using a calcium phosphate method as described previously.15 The pEGFP-C2 plasmid served as a control. Samples were fixed at 12, 24, 36 and 48 hr after transfection and mounted onto a glass microscope slide using FluorSave reagent (CalBiochem, San Diego, CA). The distributions of GFP fusion proteins were then determined using the TCS confocal system (Leica, Heidelberg, Germany). To address the variability of the fluorescence signal among the cells, we measured the relative signal intensity in nucleus vs. cytoplasm using the Image Pro Plus 4.1 software (Media Cybernetics, Silver Spring, MD). Briefly, fluorescence intensities in equally sized areas from 3 randomly selected regions in both the nucleus and cytoplasm were quantified and the relative intensity was then calculated as the ratio of the average intensity in the nucleus vs. cytoplasm. Statistical analysis was performed using ANOVA with data from 18 cells in each group followed by a t-test. The cellular localization of the GFP fusion protein in the 3 constructs and a control is shown in Figure 1. We found that the wild-type BACH1-GFP fusion protein showed typical nuclear localization, although a faint cytoplasmic staining was visible in some transfected cells (Fig. 1a). BACH1-ΔNLS1 had a typical cytoplasmic localization and the translocation of BACH1 to nucleus was almost completely blocked (Fig. 1c), indicating the importance of NLS1 in this process. In the BACH1-517T transfected cells, nuclear translocation was impaired as considerable amounts of the GFP fusion protein were retained in the cytoplasm compared to the wild-type transfectants (Fig. 1b). The relative fluorescence intensity (Table I) was significantly higher in cells transfected with the wild-type BACH1 than with the BACH1-517T or BACH1-ΔNLS1 constructs. These data suggested that the BACH1 517C→T (R173C) transition impairs the nuclear translocation but to a lesser extent than ΔNLS1. We next extended our previous genotypic analysis of the 517C→T polymorphism10 using an additional 91 normal controls and 287 breast cancer cases from the Czech population, including 136 cases with infiltrative lobular breast cancer, totalling to a sample size of 765 cases with breast cancer and 406 controls. The ascertainment of both controls and cases was as described.15 Briefly, blood samples from 151 patients were obtained from consecutive and unselected cases of breast cancer, while DNA from infiltrative lobular breast cancer carriers was extracted from the paraffin-embedded blocks surrounding cancerous tissue that were selected from a large cohort of breast cancer patients diagnosed from 1992–2000. Controls were randomly selected female blood donors from the same attraction region.15 We found 1 unaffected and 3 affected cases with the 517T/C genotype, indicating the presence of this polymorphism in a different population and a similar allelic frequency in both groups (0.3% vs. 0.5%). The combined data indicated the presence of the 517T allele in 6 of 765 (0.39%) cases and 3 of 406 (0.37%) controls in Caucasians. We also investigated whether there was any allelic imbalance in tumor DNA harbouring this polymorphism. Three samples with both germline and tumor DNA were available and were analysed by PCR-SSCP as described previously.10 The rare 517T allele was retained in tumors of all 3 cases and no obvious allelic imbalance or loss of heterozygosity was observed in any of these samples (data not shown). In summary, we confirmed that BACH1 is a nuclear protein and showed that the bipartite NLS1 was critical for its translocation to the nucleus. The nuclear transport was impaired by a single amino acid substitution in the BACH1 NLS1 resulting from a rare polymorphism in a Caucasoid population. Additional association studies in independent populations may help determine the putative role of this functional polymorphism in breast cancer susceptibility. Yours sincerely, Haixin Lei and Igor Vorechovsky We thank Drs. A. Miranda Vizuete, T. Damdimopoulos and W. Zhang for helpful discussions and Drs. J. Zaloudik and E. Jandakova for their help with samples. Lei Haixin* , Igor Vorechovsky , * Karolinska Institute, Department of Biosciences at Novum, Center for Biotechnology, Huddinge, Sweden, University of Southampton, Division of Human Genetics, School of Medicine, Southampton, United Kingdom." @default.
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• W2093354896 date "2003-02-04" @default.
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• W2093354896 title "BACH1 517C?T transition impairs protein translocation to nucleus: A role in breast cancer susceptibility?" @default.
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• W2093354896 doi "https://doi.org/10.1002/ijc.10947" @default.
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