DNA repair capacity is impaired in healthy BRCA1 heterozygous mutation carriers
Breast Cancer Research and Treatment
Tereza Vaclová1, Gonzalo Gómez-López2, Fernando Setién3, José María García Bueno4, José Antonio
Macías5, Alicia Barroso1, Miguel Urioste6, Manel Esteller3,7,8, Javier Benítez1,9,10, Ana Osorio1,9,*
1
Human Genetics Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre
(CNIO), Madrid, 28029, Spain
2
Bioinformatics Unit, Structural Biology and Biocomputing Programme, Spanish National Cancer
Research Centre (CNIO), Madrid, 28029, Spain
3
Cancer Epigenetics Group, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical
Biomedical Research Institute (IDIBELL), Barcelona, 08908, Spain
4
Medical Oncology Section, Complejo Hospitalario Universitario de Albacete, Albacete, 02006, Spain
5
Hereditary Cancer Unit, Medical Oncology Service, Hospital Morales Meseguer, Murcia, 30008, Spain
6
Familial Cancer Unit, Human Cancer Genetics Programme, Spanish National Cancer Research Centre
(CNIO), Madrid, 28029, Spain
7
Department of Physiological Sciences II, School of Medicine, University of Barcelona, Barcelona,
08907, Spain
8
Institucio Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, 08010, Spain
9
Spanish Network on Rare Diseases (CIBERER), Madrid, 28029, Spain
10
Genotyping Unit (CEGEN), Human Cancer Genetics Programme, Spanish National Cancer Research
Centre (CNIO), Madrid, 28029, Spain
*corresponding author
aosorio@cnio.es
Online Resource 1
SUPPLEMENTARY MATERIALS AND METHODS
Cell culture
Lymphoblastoid cell lines were cultured in RPMI-1640 media (Sigma-Aldrich) supplemented
with non-heat-inactivated 20% fetal bovine serum (Sigma-Aldrich), penicillin-streptomycin
(Gibco) and Fungizone (Gibco). Cell culture was carried out in 25 cm2 flasks (Corning) at 37°C in
5% CO2 atmosphere and lymphoblastoid cell lines were maintained in exponential growth by
daily dilution to 106 cells per ml of full media.
Real-time PCR analysis
The
oligonucleotides
F:
5´-TTAAAGAAAGAAAAATGCTGA-3’
and
R:
5´GGTGGTTTCTTCCATTGACC-3’ were used in the quantitative RT-PCR to detect BRCA1
expression. Normalization was carried out using expression levels of HPRT1 and MRLP19 as
internal controls. Three independent experiments were performed and relative expression was
calculated using a comparative Ct method.
Western blot analysis
Exponentially growing lymphoblastoid cell lines were harvested by centrifugation and washed
twice with ice-cold PBS (Lonza). Protein subcellular fractionation was used to detect BRCA1.
Briefly, washed cellular pellet was resuspended in RSB buffer (Tris 10 mM pH 7.5, NaCl 10 mM,
MgCl2 3 mM, protease inhibitor) and incubated for 20 minutes on ice. Nuclei were sedimented
by centrifugation, washed three times with RSB buffer, resuspended in NB buffer (Tris 10 mM
pH 7.5, NaCl 0.4 mM, EDTA 1 mM, protease inhibitor) and incubated for 15 minutes at 4°C
while shaking. Samples were then centrifuged and the concentration of the nuclear fraction of
proteins was determined using the DC Protein Assay Kit II (Bio-Rad) with BSA as the standard.
Whole cell lysates were used to detect RAD51. Cellular pellets were washed twice with ice-cold
PBS and lysed in RIPA buffer (Sigma-Aldrich) containing protease inhibitor (Roche) for 1 hour
on ice. Lysates were centrifuged at 16,000 g for 20 minutes at 4°C and supernatants were
transferred into new tubes for further quantification of proteins using the Bio-Rad protein
assay kit, as for nuclear proteins.
Seventy-five micrograms of nuclear proteins or proteins from whole cell lysate were loaded
per well of 3–8% Tris-Acetate or 4–12% Bis-Tris gel (Invitrogen), respectively. Separated
proteins were transferred to a nitrocellulose membrane (Whatman) using the NuPage transfer
system (Invitrogen) and the membrane was then blocked in Tris-buffered saline containing
0.05% Tween-20 (TBS-T) and 5% milk. Blots were probed with mouse anti-BRCA1 (Calbiochem;
#OP92) or rabbit anti-RAD51 (Santa Cruz; #sc-8349). Mouse anti-β-actin (Sigma; #A5441) and
mouse anti-HSC70/HSP70 antibodies (Enzo; #ADI-SPA-820-D) served as loading controls. The
secondary antibodies were HRP-conjugated (Dako) and the immunoblots were developed
using the ECL system (GE Healthcare). Western blot X-ray films (Kodak) were scanned and
analyzed using ImageJ software.
Protein dephosphorylation by Calf Intestinal Alkaline Phosphatase (CIP)
Nuclear proteins were isolated from exponencially growing cells as previously described in the
“Western blot analysis“ section. Samples were incubated with 1 unit of CIP/μg protein at 37°C
for 60min, separated by SDS-PAGE and transferred to a nitrocellulose membrane.
Dephosphorylation of BRCA1 by Calf Intestinal Alkaline Phosphatase was checked using mouse
monoclonal anti-BRCA1 antibody (Calbiochem, #OP92).
DNA double-strand break repair evaluation
DNA damage was induced by exposure to ionizing radiation (MDS Nordion Gammacell 1000).
Control and 10-Gy-irradiated lymphoblastoid cell lines were cultured 4 hours before fixation
with 4% paraformaldehyde (Aname). Two hours before fixation, cells were counted and
seeded into a poly-L-lysine-coated (Sigma-Aldrich) µCLEAR bottom 96-well plate (Greiner BioOne) at a density of 75,000 cells per 100ul full media per well. Lymphoblastoid cell lines were
then left for 2 hours in order to attach to the surface of the wells, fixed for 15 min at room
temperature, permeabilized in 0.5% Triton X-100 in PBS for 20 minutes at 4°C and stained with
primary and secondary antibodies and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI)
to visualize nuclei. To detect gamma-H2AX we used mouse monoclonal anti-phospho-histone
H2AX antibody (Millipore; #05-636), and to detect RAD51 a rabbit polyclonal anti-RAD51
antibody was utilized (Santa Cruz Biotechnology; #sc-8349). Alexa Fluor 488- and Alexa Fluor
568-conjugated secondary antibodies from molecular probes (Invitrogen; #A-11034 and #A11031, respectively) were used, and fluorescent images were automatically taken for each well
of the 96-well plate using an Opera High-Content Screening System (Perkin Elmer). Pictures
were taken under non-saturating conditions using a 40x magnification lens and both the
intensity of gamma-H2AX nuclear signal and the number of RAD51 foci in nuclei were
calculated.
mRNA expression profiling
100 ng of total RNA from lymphoblastoid cell lines was labeled using the Low Input Quick Amp
Labeling Kit (Agilent Technologies) following manufacturer´s instructions and the One-Color
Microarray-Based Gene Expression Analysis approach. Labeled samples were purified with
silica-based RNeasy spin columns (Qiagen) and 600 ng of labeled extract from a total volume of
50 µl was hybridized at 65°C for 17 hours. Images were scanned on a G2505C DNA microarray
scanner (Agilent) and data were extracted with Feature Extraction software (Agilent; version
10.7). Background correction (via the normexp method) and between-arraynormalization (via
the quantiles method) was performed using the Babelomics 4.3 web tool (Medina and
Carbonell et al, 2010; available at http://babelomics.bioinfo.cipf.es). The microarray dataset
has been deposited in the Gene Expression Omnibus (GEO) public repository with GEO
accession number GSE60396.
miRNA expression profiling
300 nanograms of total RNA was labeled with Hy3 fluorescent dye using the miRCURY LNA™
microRNA Hi-Power Labeling Kit (Exiqon). Labeled RNA was then hybridized onto miRNA
microarray slides for 16 hours at 56°C using Agilent SureHyb hybridization chambers and a
hybridization oven with rotation. After washing and drying, the arrays were scanned and the
images were analyzed using Feature Extraction software (Agilent; version 10.7), following a
modified Exiqon protocol. The microarray background correction was performed using the
normexp method and normalization between arrays was carried out by applying the quantiles
method in R statistical software (version 2.13.0). The microarray dataset is publicly available at
the GEO public repository (GEO accession number GSE60444).
Microarray data analysis
Normalized measures from replicate probes data were merged by taking their mean. mRNAs
and miRNAs with low variation in expression across samples (VAR < 1x10-9 and VAR < 0.002,
respectively) were excluded. This data preprocessing was performed using the preP application
available in the Asterias web server (http://asterias.bioinfo.cnio.es/). To identify differentially
expressed genes and miRNAs between control and mutated lymphoblastoid cell lines we used
the POMELO II web tool, which is also accessible via the Asterias web server. Multiple testing
was corrected for using Benjamini and Hochberg false discovery rate (FDR) adjustment. mRNAs
with FDR < 0.15 or |logFC| > 2 were considered significantly differentially expressed, whereas
FDR < 0.05 or |logFC| > 2 was used to define differentially expressed miRNAs. In order to
visualize gene expression patterns of differentially expressed genes, a heat map was created
using
GENE-E
software
version
3.0.204
(Broad
Institute;
http://www.broadinstitute.org/cancer/software/GENE-E/index.html). Pathway analysis was
performed using the Ingenuity Pathway Analysis software package (IPA;
http://www.ingenuity.com). To determine the biological functions most strongly associated
with gene expression profiles, a core analysis was performed from which the most dramatically
affected functions/pathways were extracted (P-value < 0.05). The p-values were calculated
using a one-sided Fisher´s exact test.
Statistical analysis
To test for differences between groups of lymphoblastoid cell lines, when analyzing BRCA1
mRNA and protein expression, intensity of the gamma-H2AX nuclear signal and number of
RAD51 foci in nuclei. For normally distributed variables and Mann-Whitney U test for variables
with a non-normal distribution, based on visual inspection of histograms; otherwise Student´s
t-test for independent samples was used. Statistical analyses were done using SPSS Statistics
software (version 17.0; IBM) and dot plots were created using GraphPad Prism (version 5.03;
GraphPad Software; www.graphpad.com).