Supplemental data
Material and Methods
Patient samples
Paraffin-embedded bone marrow specimens from archived stocks in the Department of Pathology,
Tokyo Medical and Dental University were used in this study. All samples had been obtained after
informed consent for laboratory use. The diagnosis of MDS was performed by clinicians with
expertise in hematology or pathology. Several samples diagnosed before 1999 were classified using
the French American British (FAB) classification (1). These samples were re-classified by updated
WHO criteria according to the numbers of blasts (2). Following this criterion, samples from refractory
anemia (RA) (10 cases), RA with excess blasts (RAEB)-I (13 cases), RAEB-II (8 cases), and OL (10
cases) were examined using immunohistochemical techniques. All samples from RA, RAEB-I and
RAEB-II were obtained at initial diagnosis. Two Bone marrow specimens from Idiopathic
Thrombocytopenia (ITP) patients and two bone marrow specimens from cancer patients without bone
marrow metastasis were included as non-malignant controls.
Antibodies
We used antibodies against Ser 1981-phosphorylated ATM clone 7C10D8 (Rockland,
Gilbertsville, PA), Thr 68-phosphorylated Chk2, -H2AX, Ser 15-phosphorylated p53 (all from Cell
Signaling, Danvers, MA), and total ATM Ab-8 (Epitomics, Inc. Burlingame, CA ), Chk2 (Vision
BioSystems, Norwell, MA), p53 Do-6 (DAKO, Glostrup, Denmark) and Ki-67 Ab-3 (Lab vision) for
immunohistochemistry, Ser 1981-phosphorylated ATM clone 10H11.E12 (Cell Signaling) and Ku70
(Santa Cruz, Santa Cruz, CA) for western blotting.
Immunohistochemistry
Indirect immunoperoxidase staining on formaldehyde-fixed, de-paraffinized tissue sections was
performed using the Vectastain Elite kit (Vector, Burlingame, CA) with DAB substrate and nickel
sulphate enhancement without nuclear counterstaining after antigen retrieval. For double staining, in
the first step, tissue was stained by Ki-67, phospho Chk2, Phospho p53, H2AX antibody (rabbit
polyclonal antibody) using Vectastain Elite ABC AP kit with Vector blue as the substrate. Following
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the stripping of Vector blue staining using ethanol, tissue was restained using phospho-ATM (mouse
monoclonal antibody) and Vectastain ABC Elite kit with DAB as the substrate. The immunostaining
patterns were evaluated by an experienced pathologist and scored as (1) negative (no positive staining
or up to 1% of scattered positive cells); (2) intermediate (heterogeneous staining, corresponding to at
least 20% of the random sections showing 2−50% positivity); or (3) high positivity (variable to almost
homogeneous staining, corresponding to at least 20% of the random sections showing 51−100%
positivity).
Immunoblotting
Bone marrow-derived mononuclear cells from control or MDS patients were kept frozen in liquid
nitrogen prior to western blotting, which was performed as previously described (3) with minor
modifications as follows: Cells were resuspended in hypotonic buffer (10 mM HEPES/KOH pH 7.5,
10 mM KCl, 1.5 mM MgCl2, 0.1% NP40, protease inhibitor and phosphatase inhibitor) and
centrifuged at 3000 x g for 3 min at 4˚C. After aspiration of cytosolic extract, nuclear extract was
obtained by 50 l high salt buffer (20 mM HEPES/KOH pH 7.5, 0.45 M NaCl, 1 mM EDTA, protease
inhibitor and phosphatase inhibitor), and centrifugation at 13,000 x g for 20 min at 4˚C. 80 g of
nuclear extract was used for immunoblotting. For detection of signals, SuperSignal West Femto
Maximum Sensitivity Substrate (PIERCE, Rockford, IL) was applied.
ATM loss of heterozygosity (LOH) analysis
Fluorescence in situ hybridization (FISH) at the chromosome 11q22.3 ATM locus and
chromosome 11 centromere was performed using LSI ATM (11q22.3) and 11CEP probes (Vysis,
Abbott Molecular Inc. Des Plaines, IL) respectively. Paraffin-embedded tissue sections were
deparaffinized and pretreated (Paraffin Pretreatment Kit II, Vysis). Hybridization was performed
according to the protocol provided by the manufacturer. Microsatellite analysis at chromosome 11q
neighboring or intragenic to the ATM locus (D11S2178, D11S1294, D11S1778, D11S2179, D11S2366,
and D11S1787) was performed as described (4). Briefly, ATM locus amplification with
fluorescent-tagged PCR primers from MDS or OL stage DNA of the same patient was performed, and
analyzed on an ABI 310 machine (Applied Biosystems, Foster City, CA). The data were processed
using the GeneScan program (Applied Biosystems).
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p53 Mutation analysis
Genomic DNA from paraffinized tissue sections was obtained using the QIAamp DNA Mini kit
(Qiagen, Hilden, Germany). Exons 5-8 of the p53 gene were amplified by PCR as previously
described (5). Direct sequence analyses of all amplifed products was performed to detect p53
mutations. To detect in-frame deletion of the p53 gene, the PCR product was TA-cloned, and 10
independent colonies were sequenced.
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