Brca1 Mutation Analysis of 41 Human Breast Cancer Cell Lines Reveals Three New Deleterious Mutants

Germ line mutations of the BRCA1 gene confer a high risk of breast cancer and ovarian cancer to female mutation carriers. The BRCA1 protein is involved in the regulation of DNA repair. How specific tumor-associated mutations affect the molecular function of BRCA1, however, awaits further elucidation. Cell lines that harbor BRCA1 gene mutations are invaluable tools for such functional studies. Up to now, the HCC1937 cell line was the only human breast cancer cell line with an identified BRCA1 mutation. In this study, we identified three other BRCA1 mutants from among 41 human breast cancer cell lines by sequencing of the complete coding sequence of BRCA1. Cell line MDA-MB-436 had the 5396 + 1G>A mutation in the splice donor site of exon 20. Cell line SUM149PT carried the 2288delT mutation and SUM1315MO2 carried the 185delAG mutation. All three mutations were accompanied by loss of the other BRCA1 allele. The 185delAG and 5396 + 1G>A mutations are both classified as pathogenic mutations. In contrast with wild-type cell lines, none of the BRCA1 mutants expressed nuclear BRCA1 proteins as detected with Ab-1 and Ab-2 anti-BRCA1 monoclonal antibodies. These three new human BRCA1 mutant cell lines thus seem to be representative breast cancer models that could aid in further unraveling of the function of BRCA1.


Introduction
Germ line mutations of the BRCA1 breast cancer susceptibility gene predispose female carriers to develop breast cancer and ovarian cancer (OMIM 113705; http://www.ncbi.nlm.nih.gov/omim/).The BRCA1 protein normally resides in a nuclear multiprotein complex, including BRCA2, BARD1, and RAD51, and the DNA damage repair proteins MSH2, MLH1, MSH6, ATM, NBS1, MRE11, RAD50, BLM, and RFC.This BRCA1-associated genome surveillance complex functions as a sensor of abnormal DNA structures, such as double-strand breaks and base pair mismatches.BRCA1 has been suggested to have a pivotal function within BRCA1associated genome surveillance complex by coordinating the actions of damage-sensing proteins and executive repair proteins.BRCA1 may also act as a transcriptional regulator of genes involved in checkpoint reinforcement and, in complexes with BARD1, as a ubiquitin ligase (reviewed in refs.[1][2][3][4].Thus, mutations of BRCA1 likely impair the repair of damaged DNA, thereby rendering the mutant cells prone to malignant transformation.To fully unravel the function of BRCA1 in DNA damage responses, cell lines with naturally occurring mutations of the gene provide invaluable research tools as they allow extensive analyses and in vitro manipulation.Only a single human BRCA1 mutant breast cancer cell line had thus far been described (HCC1937; ref. 5).To identify additional mutants, we screened 41 human breast cancer cell lines for alterations in the BRCA1 gene sequence.

Materials and Methods
Breast cancer cell lines.The 41 human breast cancer cell lines used in this study are listed in Table 1.The SUM series were generated in the Ethier laboratory (available at http://www.asterand.com).Cell lines EVSA-T, MPE600, and SK-BR-5/7 were kind gifts of Dr. N. DeVleeschouwer (Institut Jules Brodet, Brussels, Belgium), Dr. H. Smith (California Pacific Medical Center, San Francisco, CA), and Dr. E. Stockert (Sloan-Kettering Institute for Cancer Research, New York, NY), respectively.Cell line OCUB-F was obtained from Riken Gene Bank (Tsukuba, Japan).All other cell lines were obtained from American Type Culture Collection (Manassas, VA).Extensive analysis of nearly 150 polymorphic microsatellite markers had shown that all cell lines are unique and monoclonal (6).
Mutation analysis.The complete coding sequence and exon-intron boundaries of BRCA1 (Genbank U14680) were analyzed for genetic alterations in all cell lines, except for SUM44PE and ZR75.30(only exons 3-7 and 11-15 were analyzed, respectively).Exons 1a to 11 and 16 to 24 were PCR amplified from genomic DNA templates and exons 12 to 15 were amplified from RNA templates, as described (7).Amplification products were then analyzed for sequence alterations with the Big Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA) using an ABI 3100 Genetic Analyzer.All unique sequence alterations were confirmed by sequencing of an independently amplified template.This approach may allow mutations to go undetected in cell lines without allelic loss, specifically deletions when analyzing DNA and truncating mutations that result in down-regulated transcripts when analyzing RNA.Allelic loss of the BRCA1 gene was determined by PCR-based microsatellite analysis as previously described (6).BRCA1 and HPRT transcripts were concurrently amplified from RNA templates, using the Qiagen Onestep reverse transcription-PCR (RT-PCR) kit and gene-specific primers.Primer sequences are available upon request.
Immunocytochemistry. Cell lines were cultured to optimal cell density in eight T162 flasks and medium was refreshed 24 hours before harvesting.Cells were harvested by scraping, washed twice with PBS, and fixed in PBS with 2% formalin for 12 to 72 hours.Cells were then washed once with PBS, resuspended in liquidized PBS with 2% agarose, and embedded in paraffin by routine diagnostic procedures.Paraffin sections (4 Am) on Starfrost microscope slides (Knittel Gla ¨ser, Braunschweig, Germany) were routinely deparaffinized and dehydrated, and epitopes were retrieved in Tris-EDTA (pH 9.0) for 30 minutes at 100jC in a microwave oven.Slides were blocked with 2% bovine serum albumin in PBS for 30 minutes at room temperature and then incubated overnight at 4jC with antibodies diluted in Normal Antibody Diluent (Scytek Laboratories, Logan, UT).Anti-BRCA1 mouse monoclonal antibodies Ab-1 (clone MS110; 1:100 or 1 Ag/mL) and Ab-2 (clone MS13; 1:320 or 0.6 Ag/mL) were both purchased from Calbiochem (Darmstadt, Germany) and isotype-matched control monoclonal antibody X0931 (1:100 or 1 Ag/mL) from Dako (Glostrup, Denmark).Slides were developed using the DakoCytomation Envision System horseradish peroxidase (3,3V -diaminobenzidine) kit, with omission of the antiperoxidase treatment.Slides were counterstained for 5 seconds with hematoxylin.Both anti-BRCA1 antibodies were titrated in two-step serial dilutions on BRCA1 wild-type cell lines.At the presumed optimal antibody dilution, both antibodies showed distinct nuclear staining and Ab-1 also gave slight cytoplasmic staining.More diluted antibodies showed only nuclear staining for both antibodies and less diluted antibodies were aspecific (examples of wild-type and mutant cell lines at several antibody dilutions are provided as Supplementary Data).

Results and Discussion
Sequencing of BRCA1 revealed 18 different alterations in the gene sequence among 41 human breast cancer cell lines (Tables 1 and 2).Alterations were presumed to be nonpathogenic polymorphisms when they were described as such in the Breast Cancer Information Core (BIC) mutation database (http://research.nhgri.nih.gov/bic/).Together, 11 BRCA1 polymorphisms were identified among 29 of the breast cancer cell lines.Three other BRCA1 variants had been described as unclassified variant in the BIC mutation database and were each detected once in the cell lines (788 + 3G>A in MDA-MB-330 and S1140G, and 5106-20A>G both in UACC812).Deleterious BRCA1 mutations were identified in four breast cancer cell lines (Tables 1 and 2).The insertion of a cytosine residue at position 5382 of BRCA1 in cell line HCC1937 had been reported (5).In cell line MDA-MB-436, we identified the 5396 + 1G>A mutation in the splice donor site of exon 20 (Fig. 1).Analysis of BRCA1 transcripts from MDA-MB-436 identified two transcript lengths.Sequencing revealed that one transcript had skipped exon 20, predicting an in-frame deletion of 28 amino acids in the encoded BRCA1 proteins, whereas the other transcript had spliced at a cryptic splice site in intron 20 (5396 + 88/89), predicting an insertion of seven amino acids encoded by intron sequences followed by a termination codon.The patient from whom MDA-MB-436 was generated had been diagnosed with adenocarcinoma of the breast at age 39 (8), an early onset that is suggestive for hereditary breast cancer.The original tumor was not available for analysis but the 5396 + 1G>A mutation has been reported 46 times in the BIC mutation database and is classified as pathogenic.In cell line SUM149PT, we identified the deletion of a thymine residue at position 2288 of BRCA1 (Fig. 1).cFrameshift and insertion mutations are indicated by the first changed codon and the number of newly encoded codons, including premature termination codon X. Predicted effect of variant 18 is based on sequence analysis from both transcript lengths (see text).bPath, pathogenic variant according the BIC mutation database; Poly, polymorphism or nonpathogenic variant according the BIC; UV, unclassified variant according the BIC; Mut, variant not previously reported but presumed mutant as it generates a frameshift with a premature termination codon in the transcripts and because the cell line does not express nuclear BRCA1 proteins.
x Number of cell lines with a particular BRCA1 variant and number of citations of the variant in the BIC mutation database by July 2005.The 2288delT mutation predicts a shift in the BRCA1 reading frame with an insertion of 12 new amino acids after codon 723 followed by a termination codon.The patient was diagnosed at age 35 years with inflammatory breast carcinoma and she had a single known second-degree relative with postmenopausal breast cancer.The 2288delT mutation was not present in the germ line of the patient as we did not detect the mutation in a DNA sample from her blood.Of note, the identity of the donor was confirmed by analysis of 10 microsatellite markers from three chromosomes, with heterozygosity ratios of >0.80 for all markers (P < 10 À7 ).The Figure 2. BRCA1 immunocytochemistry in BRCA1 mutant and wild-type breast cancer cell lines.In contrast with the two wild-type cell lines (BT20 and SK-BR-7), none of the four BRCA1 mutants had nuclear BRCA1 staining with either of the two anti-BRCA1 monoclonal antibodies Ab-1 and Ab-2.There is some cytoplasmic staining of unclear significance in all samples with Ab-1, which is not observed with more diluted Ab-1 antibodies nor with Ab-2 (see also Supplementary Data).The negative control antibody is an IgG1 isotype-matched antibody.Magnification, Â40.

Cancer Research
Cancer Res 2006; 66: (1).January 1, 2006 original tumor was not available for analysis but the 2288delT mutation was detected in all available passages of the SUM149 cell line.It is thus unclear whether the mutation was somatically acquired during tumorigenesis in the patient or in vitro during establishment or propagation of the SUM149 cell line.Importantly, we detected the 2288delT mutation in the earliest available passage P16 and cells were only distributed to other laboratories after this passage.We identified an AG dinucleotide deletion at position 185 of BRCA1 in cell line SUM1315MO2, predicting a shift in the BRCA1 reading frame with an insertion of 16 new amino acids after codon 22 followed by a termination codon (Fig. 1).The patient was diagnosed with invasive ductal carcinoma of the breast but the age at diagnosis nor the cancer history of her family is known.The original tumor was not available for analysis but the 185delAG mutation is a well-described pathogenic BRCA1 mutation that is prevalent in the Ashkenazi Jewish population (http://research.nhgri.nih.gov/bic/).
Allelic loss of the BRCA1 gene was determined by PCR amplification of microsatellite markers D17S1321, D17S932, D17S855, D17S1327, and D17S1325.These markers are located within a 0.7 Mb chromosomal region encompassing the BRCA1 gene at 17q21.Analysis of the markers on germ line DNAs from 25 randomly selected Dutch individuals revealed heterozygosity ratios of 0.61, 0.76, 0.60, 0.55, and 0.88, respectively.Allelic loss of the BRCA1 locus was presumed when each of the five markers had a single allele size, resulting in a reliability of P = 0.002 (6).None of the 25 control DNAs had a homozygous allele pattern at the BRCA1 locus, thus validating this statistical approach.Of the 41 breast cancer cell lines, 28 (68%) had allelic loss of the BRCA1 locus, including the four BRCA1 mutants (Table 1).Similar allelic loss frequencies have been reported for primary breast cancer specimens (9)(10)(11).It is important to note that several regions at 17q are frequently amplified in human breast cancers.Allelic losses at 17q are therefore often underestimated, as karyotype-based methods do not detect loss when the retained allele is amplified or reduplicated (6,12).Indeed, we did not identify loss of the BRCA1 locus in three BRCA1 mutant cell lines that we analyzed by array comparative genome hybridization (data not shown), whereas our microsatellite analysis revealed allelic loss in all of them.Conclusively, all BRCA1 mutants were homozygous in the sequence analysis (Fig. 1).
BRCA1 transcript expression was analyzed by semiquantitative RT-PCR using five overlapping PCR fragments (Table 1).Cell lines HCC1937 and MDA-MB-436 had BRCA1 transcript expression levels that were comparable with those of most other cell lines, SUM149PT had variable but always lower expression levels, and SUM1315MO2 had consistently low expression of BRCA1 transcripts.In contrast with wild-type cell lines, nuclear BRCA1 protein expression was not detectable in any of the four mutant cell lines, as determined by immunocytochemistry on paraffin-embedded cells using anti-BRCA1 monoclonal antibodies Ab-1/MS110 and Ab-2/MS13 (Fig. 2; Supplementary Data).
We thus describe four cell lines with a BRCA1 mutation from among 41 human breast cancer cell lines, three of which had not previously been reported.All BRCA1 mutants had lost the other BRCA1 allele, in accordance with the tumor suppressor function of BRCA1.Three mutations generated a premature termination codon in the BRCA1 transcript, whereas the fourth mutation resulted in two transcripts of which one had an in-frame deletion and the other generated a premature termination codon.Three of the BRCA1 mutations have been classified as pathogenic mutations and none of the BRCA1 mutant cell lines expressed nuclear BRCA1 proteins.In an ongoing effort to characterize our panel of breast cancer cell lines, we identified biallelic mutations of the p53 tumor suppressor gene in each of the four BRCA1 mutants, 5 consistent with the notion that BRCA1 mutant tumors frequently harbor p53 mutations (reviewed in ref. 13).Pending further mutational data, these BRCA1 mutant breast cancer cell lines already are a valuable asset in pinpointing the BRCA1 functions that are critical in the suppression of breast tumorigenesis.

Figure 1 .
Figure 1.Identification of three new BRCA1 mutant breast cancer cell lines by PCR amplification and direct sequencing.Top, electropherograms displaying the wild-type sequence.Bottom, electropherograms displaying the mutations.