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SUPPLEMENTAL MATERIALS AND METHODS
Measurement of transcriptional activity. For RNA slot-blot analysis, total RNA was
extracted from untreated BMPCs and PBMCs with Trizol (Invitrogen), slot-blotted on
Hybond-N+ membrane (Amersham), hybridized to the appropriate
32P-labeled
probe and
expression of the genes of interest was evaluated by densitometric analysis (Souliotis et
al, 2006). The N-ras specific probe was a 112-bp fragment (forward primer, 5’-GTT-ATAGAT-GGTGAA-ACC-TG-3’; reverse primer, 5’-ATA-CAC-AGA-GGA-AGC-CTT-CG-3’).
The p53 specific probe was a 610-bp fragment of the human gene, and was amplified
using the following primer pair: forward primer, 5’-AGG-TTG-GCT-CTG-ACT-GTA-CC-3’;
reverse primer, 5’-ATT-GTC-CTG-CTT-GCT-TAC-CTC-3’. The d-globin specific probe was
a 204-bp PCR-fragment (forward primer, 5’-TTG-GAC-CCA-GAG-GTTCTT-TG-3’; reverse
primer, 5’-CCT-GAA-GTT-CTC-AGG-ATC-CAC-3’). Finally, the b-actin specific probe
(loading control) was a 202-bp PCR fragment (forward primer, 5’CAC-ACT-GTG-CCCATC-TAC-GA-3’; reverse, 5’-CCATCT- CTT-GCT-CGA-AGT-CC-3’).
Micrococcal nuclease (MN) digestion-based analysis. Isolation of nuclei from BMPCs
and PBMCs, digestion with MN and analysis of chromatin condensation using Southern
blot were performed as described previously (Episkopou et al, 2011). Briefly, BMPCs and
PBMCs suspension was swollen in hypotonic buffer (10mM Tris-HCl, pH 8.0, 10mM NaCl,
5mM MgCl2) for 30min on ice. After centrifugation (1400xg, 2min) the cells were
suspended in hypotonic buffer and homogenized in the presence of 0.3% Nonidet P-40.
Nuclei were purified by centrifugation (1500xg, 10min) through hypotonic buffer containing
8.5% sucrose (w/v) and resuspended in 500μl digestion buffer (100mM Tris-HCl, pH 8.0,
50mM NaCl, 3mM MgCl2, 1mM CaCl2). The isolated nuclei were immediately digested with
micrococcal nuclease (1U) for 1min at 37οC. The digestion was stopped by adding an
equal volume of stop solution (200mM Tris-HCl, pH 8.0, 200mM NaCl, 20mM EDTA, 2%
2
SDS, 200μg/ml proteinase K). The control was an undigested, freshly lysed sample of
nuclei. Genomic DNA was purified and then separated by electrophoresis in 1.5% agarose
gels. After staining with ethidium bromide, the separated DNA was transferred to
nitrocellulose and subsequently hybridized to
32P-labeled
probes specific for the genes
under study.
Measurement of total DNA damage (N-alkylpurines) along the genes. BMPCs and
PBMCs were treated ex vivo with 100μg/ml melphalan for 5min at 37oC in RPMI-1640
medium supplemented with 10% fetal bovine serum (FBS), 100units/ml penicillin,
100μg/ml streptomycin, and 2mmol/l L-glutamine, followed by incubation in drug-free
medium for 1h and DNA damage was analyzed thereafter. DNA damage (N-alkylpurines)
was measured along the Transcribed Strand (TS) and Non-Transcribed Strand (NTS) of
the N-ras, p53 and d-globin genes using Southern blot analysis (Supplementary Table 1)
(Episkopou et al, 2011). Briefly, for the measurement of melphalan-induced N-alkylpurines
(monoadducts and interstrand cross-links together), genomic DNA was fully digested with
BstYI and heated to 70oC for 30min to depurinate N-alkylated bases. Apurinic sites were
subsequently converted to single-strand breaks by the addition of NaOH for 30min at
37oC, size fractionated using agarose gel electrophoresis and Southern blotted.
Hybridizations were performed as described previously (Episkopou et al, 2011). The
N-
ras probes employed were specific for five fragments of the gene (FN1-FN5)
(Supplementary Table 1; Figure 1C). Fragment FN1 is upstream to the transcription start
site of the gene, FN2 consists of a region of 1101 nucleotides located upstream to the
transcription start site and a region of 580 nucleotides inside the gene while fragments
FN3-FN5 are located entirely inside the gene. In the case of p53, the kinetics of melphalan
adduct formation/repair were evaluated along four fragments (FP5-FP8) which are all
located inside the gene (Supplementary Table 1, Figure 1C). The d-globin probes are
specific for 4 fragments. Two of them, FD1 and FD4, are located outside the gene,
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upstream and downstream of the coding regions, respectively. Fragment FD2 contains a
region of 893 base pairs upstream of the transcription start site and the first 268 base pairs
of the coding region while the entire fragment FD3 is located inside the gene
(Supplementary Table 1, Figure 1C). In order to minimize the inaccuracy in the
measurement of DNA damage arising from errors in DNA quantitation or gel loading, in all
such experiments an internal standard (part of the N-ras gene) was included
(Supplementary Table 1). Labeling of the oligonucleotides was performed as described
previously (Episkopou et al, 2011). In brief, 0.5μg oligonucleotide dissolved in H2O, 3μl 10x
kinase buffer (0.5M Tris pH 7.6, 0.1M MgCl2, 50mM DTT), 2μl [γ-32P]ATP (7000Ci/mmole;
259TBq/mmol) and H2O were mixed so that the final volume is 30μl. Then, 25units T4
polynucleotide kinase were added and incubated for 60min at 37oC. Labeled
oligonucleotide
was
purified
away
from
unincorporated
ATP.
Purified
labeled
oligonucleotide was recovered and radioactive incorporation was then counted.
Following exposure, films were scanned using a molecular dynamics personal
densitometer and band volumes were determined. The average frequency of Nalkylpurines in the restriction fragment of interest was calculated from the fraction of DNA
in the band from the treated sample as compared to that from the non-treated control
sample. Assuming random introduction of N-alkylpurines in a homogeneous population of
fragments, we applied the Poisson expression to calculate the average number of Nalkylpurines per restriction fragment: (N-alkylpurines/fragment) = - loge(fraction of fragment
free of N-alkylpurines) (Episkopou et al, 2011).
Accumulation of p53 phosphorylated at serine15 (S15P-p53). BMPCs and PBMCs
were treated with 0-200μg/ml melphalan for 5min and then incubated in drug-free medium
for 6h. After lysis, total proteins were subjected to SDS-PAGE and Western blot analysis
as described previously (Chauhan et al, 2011). Cells were washed twice with ice-cold PBS
and lysed in 50μl lysis buffer [137mM NaCl, 20mM Tris/HCl pH 7.4, 50mM HEPES, 5mM
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EDTA, 1mM DTT, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 200mM Na3VO4, 0.5mM
PMSF, 5μg/ml aprotinin, 5μg/ml pepstatin and 50μg/ml leupeptin] followed by SDS-PAGE.
Subsequently, the proteins were transferred to nitrocellulose membranes which were
incubated for 40min at room temperature with non-fat dry milk (5%) in TBS followed by
incubation
with
the
specific
primary
antibodies
(anti-p53-phosphoS15
antibody,
abcam/ab38497 and anti-b-actin antibody, loading control, abcam/ab8227) overnight at
4oC. Then, the membranes were washed with 0.3% BSA in TBS and incubated with
horseradish peroxidase-conjugated secondary antibodies for 2h at room temperature.
After washing with TBS, the bound antibody complexes were visualized using the Western
Blotting Detection Kit ECL Plus (Amersham) and XOMAT-AR film (Kodak). The lowest
concentration of melphalan required to induce detectable levels of S15P-p53 was noted.
Apoptotic assay. Apoptosis was evaluated by using the Cell Death Detection ELISAPLUS kit (Roche Applied Sciences), according to manufacturer’s instructions. In brief,
BMPCs and PBMCs were treated with various doses of melphalan (0-200μg/ml) for 5min
followed by incubation in drug-free medium for 24h. Then, cells were collected to prepare
the cytosolic fractions that contained fragments of DNA. Equal volumes of these cytosolic
fractions were incubated in anti-histone antibody-coated wells (96-well plates), and the
histones of the DNA fragments were allowed to bind to the anti-histone antibodies. The
peroxidase-labeled mouse monoclonal DNA antibodies were used to localize and detect
the
bound
fragmented
DNA
using photometric detection with
2,29-azino-di-(3-
ethylbenzthiazoline sulfonate) as the substrate. The test quantifies apoptosis as the fold
increase (expressed as enrichment factor, EF) in the level of apoptosis in treated samples
to untreated samples. That is, we calculated the specific enrichment of mono- and oligonucleosomes released into the cytoplasm using the following formula: (EF) = (absorbance
of the treated cells) / (absorbance of the cells without melphalan treatment). The lowest
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concentration of melphalan required to induce detectable change in the levels of apoptosis
(defined as a 3fold increase relative to controls) was noted.
Supplementary Figure Legend
Supplementary Figure 1. Region-specific repair of melphalan-induced damage in the
active and silent d-globin gene in PBMCs. Box plots showing the statistical distribution
of melphalan-induced N-alkylpurine levels in healthy volunteers (HV) and MM patients,
responders (R) and non-responders (NR) to melphalan therapy, in different regions of the
TS (A, C) and NTS (B, D) of the active d-globin gene in 68 out of 85 MM patients (A, B),
and the silent d-globin gene in 17 out of 85 MM patients (C, D). The horizontal lines within
the boxes represent the median value and the vertical lines extending above and below
the boxes indicate maximum and minimum values, respectively. Correlation between DNA
damage in malignant BMPCs and DNA damage in PBMCs from the same patients in the
d-globin (E) gene is presented.