Abstract

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 low-risk 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 CD34+ hematopoietic precursors (10,13,20), others reporting a higher damage to the differentiated CD34 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 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 alkali-labile 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 A+ (erythroid) and glycophorin A (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.

RESULTS

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 A+ and glycophorin A cells, respectively (Fig. 1). Statistical analysis revealed no significant difference between the analyzed fractions of bone marrow.

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.

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.

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).

Figure 2.

Relationship between the number of erythrocytes (leukocytes) and DNA migration in glycophorin A+ (glycophorin A) 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.

Figure 2.

Relationship between the number of erythrocytes (leukocytes) and DNA migration in glycophorin A+ (glycophorin A) 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.

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 Survival (months) WBC (×109/l) Ery (×1012/l) Hb (g/l) Tr (×106/l) Neu (x 109/l) Rtc (×1012/l) Karyotype 
14 F/73  5.6 3.98 109 999 2.5 0.09 46 XX 
16 F/72 71 13.1 4.02 114 686 8.8 0.07 46 XX 
17 F/70 6 (RAEBt)a 2.79 75 34 1.4 0.07 46 XX 
19 F/67  2.52 70 145 0.03 46 XX 
20 M/74  5.1 3.08 89 198 2.5 NA NA 
28 F/71 26 2.99 83.4 187 1.8 0.02 46 XX 
34 M/35  7.9 3.35 104 482 3.2 0.07 46 XY 
35 F/66  2.4 3.31 101 219 0.6 0.04 46 XX 
P10 M/71 11 (AML)a 1.8 2.25 77 63 0.7 0.01 46 XY 
P13 M/84  3.8 3.31 100 348 1.7 0.09 46 XY 
P26 M/62  4.4 3.93 122 124 1.6 0.038 No mitoses 
P27 F/69  7.3 3.16 113 321 5.5 0.123 46 XX 
P28 M/59  3.5 3.01 101 80 1.5 0.076 46 XY 
P29 F/80  5.6 2.46 90 212 3.9 0.033 No mitoses 
Code of patient Sex/age Survival (months) WBC (×109/l) Ery (×1012/l) Hb (g/l) Tr (×106/l) Neu (x 109/l) Rtc (×1012/l) Karyotype 
14 F/73  5.6 3.98 109 999 2.5 0.09 46 XX 
16 F/72 71 13.1 4.02 114 686 8.8 0.07 46 XX 
17 F/70 6 (RAEBt)a 2.79 75 34 1.4 0.07 46 XX 
19 F/67  2.52 70 145 0.03 46 XX 
20 M/74  5.1 3.08 89 198 2.5 NA NA 
28 F/71 26 2.99 83.4 187 1.8 0.02 46 XX 
34 M/35  7.9 3.35 104 482 3.2 0.07 46 XY 
35 F/66  2.4 3.31 101 219 0.6 0.04 46 XX 
P10 M/71 11 (AML)a 1.8 2.25 77 63 0.7 0.01 46 XY 
P13 M/84  3.8 3.31 100 348 1.7 0.09 46 XY 
P26 M/62  4.4 3.93 122 124 1.6 0.038 No mitoses 
P27 F/69  7.3 3.16 113 321 5.5 0.123 46 XX 
P28 M/59  3.5 3.01 101 80 1.5 0.076 46 XY 
P29 F/80  5.6 2.46 90 212 3.9 0.033 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.

aDiagnosis at the time of death, patients 16 and 28 did not die in consequence of MDS, the other patients are still alive.

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 A cell population.

DISCUSSION

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 CD34+ bone marrow progenitors (10,13,20). In our experiments, these cells were contained in the glycophorin A fraction and thus were not separately examined. Nevertheless, our results in the glycophorin A+ 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 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 34+ 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 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

Bone marrow aspirates were diluted with Iscove’s Modified Dulbecco’s Medium (IMDM—Sigma, Germany) and kept at 4°C 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).

Table 2.

Characteristics of patients diagnosed as refractory anemia (RA) at the time of DNA fragility assessment

Code of patient Sex/age Survival (months) WBC (×109/l) Ery (×1012/l) Hb (g/l) Tr (×106/l) Neu (×109/l) Rtc (×1012/l) Karyotype 
18 F/47  3.1 1.76 77 78 0.62 0.03 47, XX, +8 
25 F/74 6 (RAEB)a 1.88 3.75 102 40 1.1 0.06 46 XX 
26 F/61  2.81 3.94 116 93 1.7 0.05 46 XX 
29 M/76  3.1 3.5 110 54 1.2 0.07 46 XY 
33 M/55 13 (AML)a 4.7 2.36 85 252 2.6 0.03 46, XY, 5q-, MCR 
37 M/61  9.8 2.01 75 437 0.04 46 XY 
39 M/66 64 2.9 2.58 96 243 1.4 <0.01 46, XY, t (2,7)(p13;p12) 
P7 M/50  3.2 3.6 116 69 1.4 0.04 46 XY 
P12 F/76  1.8 2.37 93 79 0.7 0.06 46 XX 
P20 M/79  2.4 3.15 93 53 0.7 0.02 No mitoses 
P11 M/72  4.3 2.89 99 343 3.3 0.062 47 XY 5q- 
P14 F/69  5.2 3.1 109 153 3.1 0.11 46 XX 
P15 F/79  2.7 2.4 83 164 1.9 0.06 46XX 
P16 M/82  3.6 3.39 99 317 1.8 0.02 46 XY 
P17 F/73 36 (AML)a 3.84 141 60 2.1 0.07 46, XX, 5q- 
P19 F/76 13 4.79 2.77 87 164 2.9 0.08 46 XX 
P22 F/56  2.9 1.89 66 70 2.1 0.04 46 XX 
P24 M/81  3.42 107 127 0.6 0.07 46 XY 
P32 M/64  3.6 2.88 106 161 1.3 0.05 46 XY 
P34 M/81  6.1 3.26 89 184 4.2 0.04 NA 
Code of patient Sex/age Survival (months) WBC (×109/l) Ery (×1012/l) Hb (g/l) Tr (×106/l) Neu (×109/l) Rtc (×1012/l) Karyotype 
18 F/47  3.1 1.76 77 78 0.62 0.03 47, XX, +8 
25 F/74 6 (RAEB)a 1.88 3.75 102 40 1.1 0.06 46 XX 
26 F/61  2.81 3.94 116 93 1.7 0.05 46 XX 
29 M/76  3.1 3.5 110 54 1.2 0.07 46 XY 
33 M/55 13 (AML)a 4.7 2.36 85 252 2.6 0.03 46, XY, 5q-, MCR 
37 M/61  9.8 2.01 75 437 0.04 46 XY 
39 M/66 64 2.9 2.58 96 243 1.4 <0.01 46, XY, t (2,7)(p13;p12) 
P7 M/50  3.2 3.6 116 69 1.4 0.04 46 XY 
P12 F/76  1.8 2.37 93 79 0.7 0.06 46 XX 
P20 M/79  2.4 3.15 93 53 0.7 0.02 No mitoses 
P11 M/72  4.3 2.89 99 343 3.3 0.062 47 XY 5q- 
P14 F/69  5.2 3.1 109 153 3.1 0.11 46 XX 
P15 F/79  2.7 2.4 83 164 1.9 0.06 46XX 
P16 M/82  3.6 3.39 99 317 1.8 0.02 46 XY 
P17 F/73 36 (AML)a 3.84 141 60 2.1 0.07 46, XX, 5q- 
P19 F/76 13 4.79 2.77 87 164 2.9 0.08 46 XX 
P22 F/56  2.9 1.89 66 70 2.1 0.04 46 XX 
P24 M/81  3.42 107 127 0.6 0.07 46 XY 
P32 M/64  3.6 2.88 106 161 1.3 0.05 46 XY 
P34 M/81  6.1 3.26 89 184 4.2 0.04 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.

aDiagnosis 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 61 MGUS 
C2 48 CML in hematological remission 
C3 73 CLL in hematological remission 
C4 68 MGUS 
C5 65 MGUS 
C6 86 NHL in complete remission 
C7 60 MGUS 
C8 75 MGUS 
C9 71 CLL in hematological remission 
C10 64 Chron. renal insufficiency 
C11 67 MGUS 
C12 60 MGUS 
C13 54 MGUS 
C14 72 MGUS 
C15 49 MGUS 
Code of Patient Gender Age Diagnosis 
C1 61 MGUS 
C2 48 CML in hematological remission 
C3 73 CLL in hematological remission 
C4 68 MGUS 
C5 65 MGUS 
C6 86 NHL in complete remission 
C7 60 MGUS 
C8 75 MGUS 
C9 71 CLL in hematological remission 
C10 64 Chron. renal insufficiency 
C11 67 MGUS 
C12 60 MGUS 
C13 54 MGUS 
C14 72 MGUS 
C15 49 MGUS 

MGUS, monoclonal gammapathy with unclear significance; CML, chronic myeloid leukemia; CLL, chronic lymphocytic leukemia; NHL, nonHodgkin lymphoma.

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 µl PBS. The erythroid fraction was magnetically labeled with Glycophorin A MicroBeads (Miltenyi Biotec, Germany) for 15 min at 4°C and further purified by a separation column (MS+ Separation Columns, Miltenyi Biotec, Germany) on an MACS separator (Miltenyi Biotec, Germany). After unlabeled glycophorin A (myeloid) cells elution, the column was removed from the magnetic field and labeled glycophorin A+ (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 A+ 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 µl–106 cells/ml) was mixed with 75 µl of 0.75% low melting point (LMP) agarose (Amresco, USA) and layered over 110 µl 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.

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.

FUNDING

The study was supported by grant from Ministry of Health of the Czech Republic (NR8265-3/2005).

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.

Conflict of Interest statement. None declared.

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