Human Molecular Genetics, 2008, Vol. 17, No. 14
doi:10.1093/hmg/ddn113
Advance Access published on April 21, 2008
2144–2149
DNA instability in low-risk myelodysplastic
syndromes: refractory anemia with or without
ring sideroblasts
Bozena Novotna1, , Radana Neuwirtova2, Magda Siskova2 and Yana Bagryantseva1
1
Department of Genetic Ecotoxicology, Institute of Experimental Medicine, v.v.i., Academy of Sciences of the Czech
Republic, Prague 4 142 20, Czech Republic and 2Hematooncology, 1st Medical Clinic, General University Hospital,
Prague 2 128 08, Czech Republic
Received January 17, 2008; Revised March 20, 2008; Accepted April 8, 2008
We tested genomic instability in patients with myelodysplastic syndrome (MDS) by the comet assay and verified the suitability of this approach as a tool for analysis of ineffective hematopoiesis in refractory anemia
(RA) and RA with ring sideroblasts (RARS). Erythroid and myeloid cell populations from bone marrow aspirates of 20 RA, 14 RARS and 15 control subjects were separated by differential expression of glycophorin A
and subjected to comet assay. The extent of DNA migration was measured in single cells (200 cells/bone
marrow fraction/subject). The results were in agreement with the concept of increased apoptosis in lowrisk MDS subtypes. The RA samples had a significantly higher DNA instability than controls in glycophorin
A positive cells, and the extent of DNA breakage correlated with the degree of cytopenia. Although RARS had
an even higher rate of genomic instability in bone marrow cells than RA, there was no clear relationship to
peripheral cytopenia. This suggests an additional DNA instability of non-apoptotic origin. Whether this
increase is associated with an increased repair of oxidative damage in DNA arising due to iron deposits in
ring sideroblasts remains to be formally proven. Comet assay provides a promising tool for the investigation
of difference between RA and RARS pathobiology.
INTRODUCTION
Ineffective hematopoiesis of patients with myelodysplastic
syndromes (MDS) has been attributed to excessive apoptosis
in the bone marrow precursors (1 – 4). Although some
authors observed an enhancement of apoptosis in the majority
of analyzed bone marrow samples regardless of particular
French –American – British (FAB) classification (5) subtype
of MDS (6 – 9), majority of reports described an inverse
relationship between the apoptosis of bone marrow cells and
the clinical stage of MDS (10 – 16) with decreasing apoptotic
levels noted during the progression toward leukemic transformation (17 – 19). Studies looking at apoptosis in early progenitor versus maturing bone marrow cells of MDS patients
are ambiguous, with some showing impairment of CD34þ
hematopoietic precursors (10,13,20), others reporting a
higher damage to the differentiated CD342 progeny (14,21),
with still others reporting a high level of cell death at all maturation stages, from blasts to mature cells (9,22).
Original identification of apoptotic cells by morphological
evaluation of bone marrow biopsies (1) has been subsequently
replaced by the labeling of externalized phosphatidylserine
with Annexin V (9,23) or demonstration of apoptotic cleavage
of DNA by DNA laddering, in situ end-labeling (ISEL) and
Tdt-mediated dUTP-nick end-labeling (TUNEL) (6,24).
Single-cell gel electrophoresis (or comet assay) represents
another approach capable of detecting DNA breakage (25 –
29). The comparison of comet assay with morphology of
TK6 human B-lymphoblast cells treated with hydrogen peroxide validated this approach as a reliable method for assessment of apoptosis (30). The large high-molecular-weight
fragments of DNA appearing during apoptosis before the
To whom correspondence should be addressed at: Department of Genetic Ecotoxicology, Institute of Experimental Medicine, v.v.i., Academy of
Sciences of the Czech Republic, Videnska 1083, Prague 4 142 20, Czech Republic. Tel: þ420 241062209; Fax: þ420 241062785; Email:
[email protected]
# The Author 2008. Published by Oxford University Press. All rights reserved.
For Permissions, please email: [email protected]
Human Molecular Genetics, 2008, Vol. 17, No. 14
proper cleavage to nucleosome oligomers were detected by the
comet assay soon after the labeling of externalized
phosphatidylserine with Annexin V (31). Further studies
revealed that the comet assay manifested a higher sensitivity
than the standard DNA flow cytometry (32) and the TUNEL
assay (33). Unlike the conventional methods used for the
detection of apoptotic DNA fragmentation, DNA migration
in the alkaline version of comet assay may also reflect the
genotoxic damage (single- or double-strand breaks and alkalilabile sites in DNA) as well as incisional nicks during
nucleotide excision repair of DNA (28).
The acquired DNA lesions and effectivity of DNA repair
could play an important role in pathogenesis and clinical
course of MDS (34 – 36). Therefore, we decided to utilize
the advantage of comet assay to detect both damage/repair
and apoptotic cleavage of DNA for investigation of DNA
instability in low-risk MDS, i.e. RA and RARS. Since the
pathologic phenotype of these MDS subtypes is predominantly restricted to the impairment of the erythroid cell
lineage, we separately examined glycophorin Aþ (erythroid)
and glycophorin A2 (myeloid) bone marrow fractions in a
group of patients with RA and RARS with the following
objectives: (i) to compare the extent of DNA damage in
the two fractions; (ii) to detect any difference between RA
and RARS and (iii) to verify whether the results of comet
assay correlate with the clinical status and laboratory data
of the patients.
2145
Figure 1. The extent of DNA migration in bone marrow cells: comparison of
analyzed groups. RA, refractory anemia; RARS, refractory anemia with ring
sideroblasts. The circles represent medians obtained from 200 cells per individual and bone marrow fraction. The horizontal lines depict an arithmetic
mean from individual medians characterizing persons within a given group.
RESULTS
DISCUSSION
Within a group of control persons, the medians of DNA
migration (Tail DNA) ranged from 1.96 to 8.84 and from
2.26 to 10.76% in glycophorin Aþ and glycophorin A2
cells, respectively (Fig. 1). Statistical analysis revealed no significant difference between the analyzed fractions of bone
marrow.
Compared with controls, the patients with RA and RARS
exhibited a high inter-individual variability (Fig. 1). In erythroid fraction, the both groups reached a higher level of
DNA migration than controls (P , 0.001). The values in
RARS patients even markedly exceeded those detected in
RA patients (P , 0.05). In contrast, only RARS patients
showed a significant DNA damage in myeloid fraction compared with controls. Nevertheless, the average levels of
DNA migration were lower than those detected in erythroid
fraction (P , 0.05). Two RARS patients (no. 14 and 19) displayed an extreme values of DNA migration in both analyzed
cell populations despite that their hematological examination
revealed only an impairment of the erythroid cell lineage
and the course of the disease appeared relatively favorable
(both are still alive; Table 1). No other patients within this
group showed any relationship between the hematological
data and the results of the comet assay (Fig. 2).
On the other hand, correlation analysis revealed a significant association between the degree of peripheral cytopenia
and the extent of DNA migration in erythroid bone marrow
cells of RA patients (Fig. 2). A similar, although less pronounced, trend was apparent in the glycophorin A2 cell population.
Our results demonstrated a significantly higher instability of
DNA in bone marrow cells of patients with low-risk MDS,
especially in erythroid fraction, compared with age-matched
controls that corresponded to the reported data on increased
apoptosis in these MDS subtypes. One could speculate, therefore, that this finding predominantly reflects the apoptotic
fragmentation of DNA. Correlation analysis between the
results of comet assay and cytopenia provided a valid argument because the RA patients clearly demonstrated an
inverse relationship between the extent of DNA migration in
erythroid bone marrow cells and the erythrocyte counts in peripheral blood.
Many investigators studying low-risk MDS detected an
increased cell death in CD34þ bone marrow progenitors
(10,13,20). In our experiments, these cells were contained in
the glycophorin A2 fraction and thus were not separately
examined. Nevertheless, our results in the glycophorin Aþ
population support the findings of others—i.e. that premature
apoptosis also affects maturing cells (9,14,21,22).
Unexpectedly, we observed no relationship between the
results of comet assay and cytopenia in the patients with
RARS although their levels of DNA fragmentation exceeded
significantly even the values detected in the patients with
RA. Hence, the DNA damage in RARS patients apparently
involved additional breaks of non-apoptotic origin. The basic
morphological feature discerning RARS from RA is the presence of ring sideroblasts with iron (Fe) deposits in the mitochondria. The high content of Fe could contribute to oxidative
stress, thus inducing oxidative damage in the DNA of bone
2146
Human Molecular Genetics, 2008, Vol. 17, No. 14
Table 1. Characteristics of patients diagnosed as refractory anemia with ring sideroblasts (RARS) at the time of DNA fragility assessment
Code of patient
Sex/age
14
16
17
19
20
28
34
35
P10
P13
P26
P27
P28
P29
F/73
F/72
F/70
F/67
M/74
F/71
M/35
F/66
M/71
M/84
M/62
F/69
M/59
F/80
Survival (months)
71
6 (RAEBt)a
26
11 (AML)a
WBC (109/l)
Ery (1012/l)
Hb (g/l)
Tr (106/l)
Neu (x 109/l)
Rtc (1012/l)
Karyotype
5.6
13.1
4
6
5.1
4
7.9
2.4
1.8
3.8
4.4
7.3
3.5
5.6
3.98
4.02
2.79
2.52
3.08
2.99
3.35
3.31
2.25
3.31
3.93
3.16
3.01
2.46
109
114
75
70
89
83.4
104
101
77
100
122
113
101
90
999
686
34
145
198
187
482
219
63
348
124
321
80
212
2.5
8.8
1.4
4
2.5
1.8
3.2
0.6
0.7
1.7
1.6
5.5
1.5
3.9
0.09
0.07
0.07
0.03
NA
0.02
0.07
0.04
0.01
0.09
0.038
0.123
0.076
0.033
46 XX
46 XX
46 XX
46 XX
NA
46 XX
46 XY
46 XX
46 XY
46 XY
No mitoses
46 XX
46 XY
No mitoses
Bold numbers designate the patients with multilineage dysplasia.
RAEBt, refractory anemia with excess of blasts in transformation; AML, acute myeloblastic leukemia; WBC, white blood cells; Ery, erythrocytes; Hb,
hemoglobin; Tr, thrombocytes; Neu, neutrophils; Rtc, reticulocytes; NA, not available.
a
Diagnosis at the time of death, patients 16 and 28 did not die in consequence of MDS, the other patients are still alive.
to the generally better prognosis of this MDS subtype compared with RA.
In conclusion, our results encourage the use of comet assay
for investigation of MDS. The method is simple, requires
minimal amount of analyzed material and detects the extent
of DNA damage at the level of single cell. In addition, the
modifications in the comet assay protocol allow to identify
the specific DNA lesions (including oxidative damage) as
well as to study the effectivity of DNA repair (28). This
could reveal the primary difference between RA and RARS
and offer new insights into the pathogenesis of these MDS
subtypes.
MATERIALS AND METHODS
Cell sampling
Figure 2. Relationship between the number of erythrocytes (leukocytes) and
DNA migration in glycophorin Aþ (glycophorin A2) bone marrow cells of
MDS patients. RA, refractory anemia; RARS, refractory anemia with ring
sideroblasts, continuous line represent the join of the trend; r, Pearson correlation coefficient; n.s., not significant.
marrow cells. It is possible that the process of ongoing repair
of oxidized nucleotides may be detected in the comet assay as
an additional increase of DNA migration.
Oxidized pyrimidine nucleotides have already been demonstrated in the progenitor CD 34þ bone marrow cells of MDS
patients (37). The capacity of DNA repair mechanisms
undoubtedly influences the capability of impaired cells to
survive, because only unrepaired or misrepaired DNA
lesions will direct the cell to the death pathway. Provided
that the oxidative DNA damage plays an important role in
the pathogenesis of RARS, its effective repair could contribute
Bone marrow aspirates were diluted with Iscove’s Modified
Dulbecco’s Medium (IMDM—Sigma, Germany) and kept at
48C until the next day (a maximum of 24 h) when the comet
assay was performed. Thirty-four patients (mean age + SD:
69 + 11 years; range, 35– 82) with low-risk MDS classified
according to FAB criteria (5) were investigated (Tables 1
and 2). The control group consisted of 15 patients (mean
age + SD: 65 + 10 years; range, 48 –86) without any disorder
of hematopoiesis (Table 3).
Informed consent was obtained from all patients, and the
study followed the guidelines of the institutional ethical committee.
Cell processing
Bone marrow samples were diluted with phosphate-buffered
saline (PBS), and mononuclear cell (MNC) fraction was isolated by density gradient centrifugation over Histopaque
1077 (Sigma, Germany) and washed with PBS. After centrifugation (400g for 10 min), the cell pellet was resuspended in
200 ml PBS. The erythroid fraction was magnetically labeled
with Glycophorin A MicroBeads (Miltenyi Biotec,
Human Molecular Genetics, 2008, Vol. 17, No. 14
2147
Table 2. Characteristics of patients diagnosed as refractory anemia (RA) at the time of DNA fragility assessment
Code of
patient
Sex/age
18
25
26
29
33
37
39
P7
P12
P20
P11
P14
P15
P16
P17
P19
P22
P24
P32
P34
F/47
F/74
F/61
M/76
M/55
M/61
M/66
M/50
F/76
M/79
M/72
F/69
F/79
M/82
F/73
F/76
F/56
M/81
M/64
M/81
Survival (months)
6 (RAEB)a
13 (AML)a
64
36 (AML)a
13
WBC (109/l)
Ery (1012/l)
Hb (g/l)
Tr (106/l)
Neu (109/l)
Rtc (1012/l)
Karyotype
3.1
1.88
2.81
3.1
4.7
9.8
2.9
3.2
1.8
2.4
4.3
5.2
2.7
3.6
6
4.79
2.9
2
3.6
6.1
1.76
3.75
3.94
3.5
2.36
2.01
2.58
3.6
2.37
3.15
2.89
3.1
2.4
3.39
3.84
2.77
1.89
3.42
2.88
3.26
77
102
116
110
85
75
96
116
93
93
99
109
83
99
141
87
66
107
106
89
78
40
93
54
252
437
243
69
79
53
343
153
164
317
60
164
70
127
161
184
0.62
1.1
1.7
1.2
2.6
7
1.4
1.4
0.7
0.7
3.3
3.1
1.9
1.8
2.1
2.9
2.1
0.6
1.3
4.2
0.03
0.06
0.05
0.07
0.03
0.04
,0.01
0.04
0.06
0.02
0.062
0.11
0.06
0.02
0.07
0.08
0.04
0.07
0.05
0.04
47, XX, þ8
46 XX
46 XX
46 XY
46, XY, 5q-, MCR
46 XY
46, XY, t (2,7)(p13;p12)
46 XY
46 XX
No mitoses
47 XY 5q46 XX
46XX
46 XY
46, XX, 5q46 XX
46 XX
46 XY
46 XY
NA
Bold numbers designate the patients with multilineage dysplasia.
RAEB, refractory anemia with excess of blasts; AML, acute myeloblastic leukemia; WBC, white blood cells; Ery, erythrocytes; Hb, hemoglobin; Tr,
thrombocytes; Neu, neutrophils; Rtc, reticulocytes; MCR, multiple chromosome rearrangements; NA, not available.
a
Diagnosis at the time of death, patients 39 and P19 did not die in consequence of MDS, the other patients are still alive.
Table 3. Characteristics of controls
Code of Patient
Gender
Age
Diagnosis
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
F
M
M
F
F
F
M
M
M
M
F
F
F
M
F
61
48
73
68
65
86
60
75
71
64
67
60
54
72
49
MGUS
CML in hematological remission
CLL in hematological remission
MGUS
MGUS
NHL in complete remission
MGUS
MGUS
CLL in hematological remission
Chron. renal insufficiency
MGUS
MGUS
MGUS
MGUS
MGUS
MGUS, monoclonal gammapathy with unclear significance; CML,
chronic myeloid leukemia; CLL, chronic lymphocytic leukemia; NHL,
nonHodgkin lymphoma.
Germany) for 15 min at 48C and further purified by a separation column (MSþ Separation Columns, Miltenyi Biotec,
Germany) on an MACS separator (Miltenyi Biotec,
Germany). After unlabeled glycophorin A2 (myeloid) cells
elution, the column was removed from the magnetic field
and labeled glycophorin Aþ (erythroid) cells were released.
The effectiveness of separation was verified in smears prepared from suspension of the erythroid or remaining myeloid
cell population. Contamination of the negative fraction by glycophorin Aþ cells did not exceed 5% and the positive fraction
exhibited a similar purity. The number of viable cells determined using 0.2% trypan blue stain exclusion did not fall
below 75% in any cell fraction of analyzed persons.
Single-cell gel electrophoresis (comet assay)
In principle, alkaline version of the comet assay as described
by Singh (25) was used for preparation of slides. Cell suspension (20 ml–106 cells/ml) was mixed with 75 ml of 0.75% low
melting point (LMP) agarose (Amresco, USA) and layered
over 110 ml of 0.75% normal melting point (NMP) agarose
attached to microscopic slide (SuperFrost Plus, Menzel
GmbH & Co KG, Germany) that was precoated with 2%
agarose. After 5 min solidification on ice, the slides were submerged for 1 h in a lyzing solution (2.5 M NaCl, 100 mM
EDTA, 10 mM Tris, 0.16 M DMSO, 0.016 mM Triton X-100,
all Sigma, USA) at pH 10. The slides were equilibrated for
40 min in alkaline buffer (0.3 M NaOH, 1 mM EDTA, pH 13)
to allow the DNA to unwind. Following this, the slides were
electrophoresed for 20 min in fresh alkaline buffer (1.2 V/
cm, 300 mA), neutralized in 0.4 M Tris (pH 7.5), fixed in
methanol (15 min), dried at room temperature and stored.
Before analysis, the slides were rehydrated in distilled water
and stained with 0.005% ethidium bromide (Sigma,
Germany) for 7 min. Images were captured with CCD
camera (VDS, Vosskühler, Germany) attached to a VANOX
BHS fluorescence microscope (Olympus, Japan). The extent
of DNA migration was quantified using Lucia G 4.81 software
(Laboratory Imaging, Czech Republic) in 200 cells per cell
fraction. The results of single measurements were expressed
as ‘Tail DNA’, representing the percentage of migrated
DNA from the total nuclear DNA.
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Human Molecular Genetics, 2008, Vol. 17, No. 14
Statistical analysis
Medians from measured values in erythroid or myeloid fraction characterizing each person were used for statistical analysis of differences between the control and study groups and
between the bone marrow fractions within analyzed groups.
Statistical analysis was performed using a non-parametric
Mann – Whitney rank-sum test. Pearson’s correlation was
used to test the relationship between the degree of DNA fragmentation in bone marrow cells and peripheral cytopenia.
12.
13.
14.
ACKNOWLEDGEMENTS
The authors thank Prof. K. Michalova and Dr. Z. Zemanova,
from the Centre of Cancer Cytogenetics, General Faculty Hospital, Prague, Czech Republic, for cytogenetic analysis of the
patients and Dr. J. Vorlicek, from the Department of Mathematics, Institute of Physiology of the Czech Academy of
Sciences, Prague, Czech Republic, for statistical consultation.
15.
Conflict of Interest statement. None declared.
17.
16.
18.
FUNDING
19.
The study was supported by grant from Ministry of Health of
the Czech Republic (NR8265-3/2005).
20.
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