Cells and cell lines
Fibroblasts from individuals heterozygous for BRCA2 999del5 were grown in RPMI 1640 (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 20% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin. Overexpressions were performed in COS7 cells (ATCC, Rockville, MD, USA) grown in DMEM (Invitrogen Life Technologies) supplemented with 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin.
RNA isolation, cDNA synthesis and cloning of BRCA2 999del5
Total RNA was isolated using the TRIZOL Reagent (Invitrogen Life Technologies). Three micrograms of total RNA were used for cDNA synthesis (First-Strand cDNA Synthesis kit; Amersham Biosciences, Hilleröd, Denmark). The open reading frame of BRCA2 999del5 was amplified using a primer pair where the 5' primer was complementary to the Kozak sequence of the BRCA2 cDNA and the 3' primer covered a 22 bp region prior to the putative stop codon of the BRCA2 999del5 cDNA. The sequences were GTAAAAATGCCTATTGGATCC for the 5' cloning primer, and AATGAATTCCCTGATGTTTTTC for the 3' cloning primer.
The reaction mixture contained 2 μl cDNA template, 2 mM MgCl2, 15 nmol dNTPs, 15 pmol each primer and 2 U Taq polymerase. The reaction proceeded at 95°C for 5 min, followed by 40 cycles of 94°C for 1 min, 55°C for 45 s and 72°C for 1 min, and finally heating to 72°C for 5 min and cooling to 4°C.
The amplified BRCA2 product was isolated by electrophoresis on a 1% agarose gel, and then the DNA purified (GFX PCR DNA and Gel Band Purification Kit; Amersham Biosciences), cloned into pCR2.1-TOPO vector (Invitrogen Life Technologies) and used to transform TOP10 bacteria. Plasmids were purified from individual bacterial clones (Qiagen Plasmid purification Kit; Qiagen GmbH, Hilden, Germany) and the clones were sequenced (ABI PRIZM Big Dye Terminator Cycle Sequencing Ready Reaction kit; Applied Biosystems, Foster City, CA, USA). After the deletion had been confirmed, the mutated DNA insert was excised from the TOPO-TA plasmid with HindIII and NotI restriction enzymes (New England Biolabs, Beverly, MA, USA) and ligated into the pIND(SP1)/V5-His expression vector (Invitrogen Life Technologies).
Transfections and activation of expression
Cells were grown to 60–65% confluency. Four microliters of FuGENE 6 reagent (Roche Applied Science, Hvidovre, Denmark) were added to 250 μl serum-free medium and the mixture was incubated for 5 min at room temperature. After the addition of plasmid DNA (2 μg per transfection), the mixture was incubated for 20 min at room temperature and then added to the cell cultures that had received fresh serum-containing medium. The cell cultures were allowed to recover after transfection for 24 hours, fresh medium was added and the cells were subsequently activated with ponasterone A (Invitrogen Life Technologies) at a concentration of 2.5 μg/ml, as recommended by the manufacturer.
Cell lysis, precipitations, and Western blotting
Cells were lysed in RIPA lysis buffer containing 1% Triton X-100, 1% deoxycholic acid, 20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 2.5 mM EDTA, 10% glycerol and 10 mM Na3P2O7, as well as 160 inhibitory units/ml aprotinin and 0.5 mM phenylmethylsulfonyl fluoride. After lysis, 10% of the lysate was removed (for use as a control) and the rest was incubated with antibody. For immunoprecipitations, 2.5 μl primary monoclonal antibody or 5 μl primary polyclonal antibody were used per precipitation. After the addition of antibody, the samples were subjected to gentle end-over-end shaking for 1–2 hours at 4°C. Subsequently, 50 μl Protein G Sepharose beads (Amersham Biosciences) were added to each sample and the mixture incubated for a further 1 hour at 4°C. The beads were washed with lysis buffer, the supernate was removed and 50 μl of 2 × sample buffer containing β-mercaptoethanol were added to the beads. The samples were boiled for 5 min and were centrifuged for 30 s.
For precipitation of the overexpressed 6 × His-tagged proteins, Ni-NTA Magnetic Agarose Beads (Qiagen GmbH) were used. For purification of the 6 × His-tagged proteins under native conditions, cells transiently transfected with pIND-BRCA2 999del5 or with pIND-LacZ were lysed in lysis buffer containing 50 mM NaH2PO4 (pH 8), 300 mM NaCl, 10 mM imidazole, 1% Tween 20. Ten microliters of 5% Ni-NTA Magnetic Agarose Bead suspension were added to the lysate and incubated for 1–2 hours at 4°C. The supernate was separated from the beads using a Qiagen 12-Tube Magnet (Qiagen) and the beads were washed several times with washing buffer containing 50 mM NaH2PO4 (pH 8), 300 mM NaCl, 20 mM imidazole, 0.05% Tween 20. The proteins were eluted from the beads with 25 μl elution buffer containing 50 mM NaH2PO4 (pH 8), 300 mM NaCl, 250 mM imidazole, 0.05% Tween 20, an equal volume of 2 × sample buffer supplemented with β-mercaptoethanol was added to the eluates, and the samples were boiled for 5 min and centrifuged for 30 s.
Purification of the 6 × His-tagged proteins under denaturing conditions proceeded in exactly the same way except that the cells were lysed in a buffer containing 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris–Cl (pH 8), that the agarose beads with the bound 6 × His-tagged proteins were washed with a buffer containing 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris–Cl (pH 6.3), and that a buffer containing 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris–Cl (pH 4.5) was used for elution of the proteins from the beads.
The precipitates and the lysates were separated by electrophoresis on gradient 6–18% polyacrylamide gels and blotted onto a Hybond-P membrane (Amersham Biosciences). The primary antibodies used in this study were: SC1 and SC3 rat monoclonal antibodies raised against the N-terminal and C-terminal sequences of BRCA2, respectively (a generous gift from D Bertwistle and A Ashworth); anti-V5 mouse monoclonal antibody (Invitrogen Life Technologies); anti-actin mouse monoclonal antibody (SDS); anti-BRCA2 rabbit polyclonal antibody raised against the putative out-of-frame residues at the C-terminus of BRCA2 999del5 (Bethyl Laboratories, Montgomery, TX, USA); and NCL-p53-DO7 mouse monoclonal antibody against p53 (Novocastra, Newcastle, UK). Secondary peroxidase-conjugated donkey anti-mouse and donkey anti-rabbit horseradish antibodies were purchased from Amersham Biosciences and peroxidase-conjugated goat anti-rat antibody was purchased from Pierce Biotechnology (Rockford, IL, USA). The ECL detection system (Amersham Biosciences) was used to produce images.
Total RNA samples (about 15 μg each) were separated by electrophoresis on a 1% agarose gel containing 2.2 M formaldehyde and 0.02 M 3-(N-morpholino)propanesulfonic acid, stained with ethidium bromide and visualized by UV. The gels were blotted onto Hybond-N+ membrane (Amersham Biosciences) and RNA was subsequently fixed by exposing the membrane to UV light for 5 min. The BRCA2 999del5 insert of the pIND-BRCA2 999del5 plasmid was excised from the vector with HindIII and NotI. Then 100 ng insert were labeled using the ECL direct nucleic acid labeling system (Amersham Biosciences) and were used as a probe. The labeled probe was added to a hybridization buffer containing 0.5 M NaCl and 5% blocking agent, and the mixture was incubated with the membrane overnight at 42°C. The images were developed using the ECL detection system (Amersham Biosciences).
F96 Maxisorp immunoplates from Nunc (Roskilde, Denmark) were coated with cell lysates. The synthetic 16 amino acid peptide alone (the predicted 16 out-of-frame amino acid stretch of BRCA2 999del5), and the same peptide conjugated to keyhole limpet hemocyanin carrier protein were used as controls. Briefly, wells were coated with different dilutions (1:10, 1:100, 1:1000) of lysates prepared from the heterozygous fibroblasts on one hand, and from homozygous wt fibroblasts on the other hand, all made in duplicate. About 250 ng synthetic peptides were used to coat each control well. All the samples were diluted in coating buffer containing 0.015 M Na2CO3 0.035 M NaHCO3, and 0.003 M NaN3 (pH 9.6), which was also used as a blank. The same experiment was performed with the lysates of the COS7 cells transfected with pIND-BRCA2 999del5 or with pIND-LacZ as control. The antibody against the putative 16 out-of-frame amino acid stretch of BRCA2 999del5 (dilution 1:2500) was used to detect the presence of the mutated BRCA2 protein. Preimmune serum was used as a control. The secondary antibody used was goat anti-rabbit alkaline phosphatase-conjugated antibody (DAKOCytomation, Glostrup, Denmark) diluted 1:3000 in PBS–0.05% Tween 20.
To investigate whether the proteasome plays a role in the instability of BRCA2 999del5 protein, the proteasome inhibitor lactacystin was used to treat cells overexpressing pIND-BRCA2 999del5 or pIND-LacZ. Briefly, 24 hours after transfection, the cells were treated with ponasterone A and consequently with lactacystin at a concentration of 10 μM for different periods of time. The cells were thereafter lysed and subjected to electrophoresis on gradient 6–18% polyacrylamide gel, transferred onto Hybond-P membrane and blotted with α-V5 or with the antibody against the nonrelevant residues of BRCA2 999del5 or SC1 antibody. Blotting with α-actin was performed as a control for protein loading, and blotting with α-p53 was performed as a control for proteasome inhibition.