- Email: [email protected]
Increased sister chromatid exchange frequency in bone marrow cells of myelodysplastic syndromes
Increased Sister Chromatid Exchange Frequency in Bone Marrow Cells of Myelodysplastic Syndromes P. Balakrishna Murthy, Nanao Kamada, and Atsushi Kuramoto
ABSTRACT: The frequency and distribution of sister chromatid exchange (SCE) was determined in bone marrow and peripheral lymphocytes of patients with preleukemic myelodysplastic syndromes. Patients with refractory anemia (RA), RA with excess of blasts (REAB), RA with ring sideroblasts, and RA in transformation presented a 2-3-fold increase in SCE frequency in the bone marrow cells. In contrast, lymphocytes from these patients showed only a marginal increase in SCE. Analysis of interchromosomal distribution of SCE indicated a preferential involvement of chromosomes in group C in patients with RA with excess of blasts. Furthermore, the SCE in patients was not found to be influenced by the karyotype status. INTRODUCTION Analysis of sister chromatid exchange (SCE) in b r o m o d e o x y u r i d i n e (BrdU) labeled chromosomes has been suggested to be a sensitive parameter of c h r o m o s o m a l instability [1,2]. Since the initial description of a manyfold increase in SCE frequency in cells from Bloom's s y n d r o m e [3], a search for a possible significance of SCE in various h u m a n states has been initiated. Studies on the a p p l i c a t i o n of SCE in h u m a n malignant and n e o p l a s i a - p r o n e situations have been a i m e d at looking into the probable diagnostic and prognostic importance of SCE in these diseases [4]. The results of these studies [4, 5], albeit controversial, have pointed to either slightly elevated or significantly r e d u c e d SCE in malignant tissues. P u b l i s h e d reports dealing with SCE in p r e l e u k e m i c m y e l o d ysplastic syndromes (MDS) have been few and have resulted in conflicting data [6, 7]. While Carbone et al. [7] have reported an elevated SCE frequency, particularly in chromosome #1, in cells from patients with refractory anemia (RA), Knuutila et al. [6] could not find such an alteration in patients with refractory i d i o p a t h i c sideroblastic anemia (SA). In view of this controversy, and in view of the fact that patients with MDS present an increased risk for acute leukemia [8], a study on the frequency of SCE in MDS becomes meaningful. We present the results of our studies on the frequency and distribution of SCE in bone marrow (BM) cells and peFrom the Department of Internal Medicine, Research Institute for Nuclear Medicine and Biology,Hiroshima University, Hiroshima, Japan. Address requests for reprints to Dr. Nanao Kamada, Department of Internal Medicine, Research Institute for Nuclear Medicine and Biology, Hiroshima University, 1-2-3 Kasumi-cho, Hirashima 734, Japan. Received December 29, 1983; accepted March 30, 1984.
151 © 1985 by Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017
Cancer Genetics and Cytogenetics15,151 158 (1985) 0165-4608/85/$03.30
15 2
P . B . Murthy et al.
ripheral l y m p h o c y t e s of patients with MDS. In addition, we attempt to analyze the frequency of SCE in relation to the BM karyotype status of the patients. MATERIALS AND METHODS
Eleven patients diagnosed as having MDS according to the French-American-British classification [9] were investigated. There were six with RA with excess of blasts (RAEB), two with RA, two with RAEB in transformation, and one with SA. Six patients with hematologically normal BM were also investigated. BM from patients prior to therapy and from controls was obtained, and mononuclear cells were separated by the Ficoll m e t h o d [10]. After Ficoll separation, the majority of the cell p o p u l a t i o n in most subjects from the two groups, was c o m p r i s e d of immature granulocytes, erythroblasts, lymphocytes, and monocytes in that order. A part of the m o n o n u c l e a r cells separated from the BM was used for direct karyotype investigation on G-banded preparations using a standard technique described previously [11], and the rest of it was used for SCE investigation, as described below. Two samples of BM cells of 3 x 105 cells each were cultured in RPMI 1640 m e d i u m s u p p l e m e n t e d with 10% fetal calf serum, L-glutamine, and antibiotics. BrdU (Sigma, St. Louis, MO) at a final concentration of 10p.M was a d d e d to the cells, and the cultures incubated at 37°C in the dark for 48 hr. Colchicine (0.05 ~g/ ml) was a d d e d during the last 1 hr of incubation. Cells were collected, hypotonically shocked (0.075 M KC1), fixed, and w a s h e d with methanol and acetic acid (3:1) and d r o p p e d onto cleaned slides. Air-dried preparations were exposed to Hoechst 33258 (0.5 ~g/ml) in p h o s p h a t e buffer (pH 7.0), exposed to ultraviolet light, and stained with 4% Giemsa [12]. Peripheral blood samples were also obtained from patients and controls at the time of collection of BM. Lymphocytes in buffy coat were cultured in RPMI 1640 m e d i u m with the same s u p p l e m e n t s as described above. Phytohemagglutinin (Wellcome) was a d d e d to cells and the cells incubated at 37°C for 24 hr. After this period, BrdU, at a final concentration of 10 p.M, was introduced into the cultures and the cells incubated for an additional 48 hr. After colchicine arrest, metaphase preparations were m a d e and stained for SCE as described above. SCEs were scored in at least 20 second-division metaphases from each BM and peripheral l y m p h o c y t e preparation. Agreement between two investigators was obtained before each SCE was scored. All metaphases scored were subsequently photographed, and karyotypes were prepared according to ISCN [13]. SCEs were identified in i n d i v i d u a l chromosomes or chromosome groups. RESULTS
Table 1 presents the clinical and hematologic data of the patients, as well as the results of the karyotype investigations. Data on the mean frequencies of SCEs are presented in Table 2. SCE frequencies in BM of six controls ranged between 2.41 and 3.76, with a mean of 3.08 ± 0.24 (mean ± SE). The corresponding mean values in patients with RAEB was 6.02 ± 0.35 (range 5.08-7.17). This difference was statistically significant. Lymphocytes from RAEB showed a mean SCE rate of 13.51 -+ 1.02, w h i c h differed significantly from that of 8.93 ± 0.48 seen in controls. The mean SCE frequencies in BM cells from two patients with RA were 6.48 ± 2.41 (mean ± SD) and 5.54 ± 1.82, each of w h i c h varied significantly with the mean level seen in control BM cells. Peripheral lymphocytes in RA showed only a marginal increase in SCE levels.
153 Table 1
C l i n i c a l , H e m a t o l o g i c , a n d K a r o t y p e D a t a of P a t i e n t s
Patients RAEB RAEB RAEB RAEB RAEB
1, 2, 3, 4, 5,
M, 69 M, 69 M, 68 F, 63 M, 54
RAEB 6, F, 74
WBC (>< 109/L)
NCC
Blasts + pro
6.0 7.2 5.7 1.6 3.7
8.7 8.6 9.5 7.1 8.5
178 42 121 150 99
Hyper Hyper Normo Hyper Normo
8.0 7.0 6.5 7.0 7.0
9.8
5.9
117
Normo
6.8
7.1 6.8
79 79
Hyper Hyper
5.7 5.1
8.1 8.7
21 135
Hyper Hyper
22.8 17.2
5.7
158
Hyper
4.0
RA 1, M, 79 2.1 RA 2, F, 82 1.9 RAEB in transformation Case 1, M, 47 1.3 Case 2, M, 78 4.1 RA w i t h ring sideroblasts Case 1, M, 66 3.5
Table 2
Hb PLts (g/dl) ( x 109/L)
Karyotype 46,XY, - 13, + 18/45,XY, - 13/46,XY 46,XY/47,XY, + 18 46,XY/45,XY,- 8 46,XX 45,XY, - 8,del(5)(q14),16q + ,17p + / 44,XY, - 8, - 9,del(5)(q14), 16q+,17p+ 47,XX,del(3)(q11),t(4;17) (p16;p13),del(5)(q13), del(12)(p12),t(12;?)(p13;?), del(15)(q22), + 17/ 46,XX,del(3)(q11), t(4;17)(p16;p13),del(5)(q13), del(12)(p12),t(12;?)(p13;?), del(15)(q22), + 17, - 20 46,XY, + 9, - 17/46,XY 46,XX
47,XY, + 8/46,XY 46,XY/46,XY,del(5)(q21) 46,XY
SCE F r e q u e n c y i n P a t i e n t s w i t h M D S BM
Subject Control RAEB RA Case 1 Case 2 RAEB in transformation Case 1 Case 2 RA w i t h ring sideroblasts
Total ceils
Total SCEs
171 148
527 891
21 26 20 (26) 25 25
Lymphocyte Mean SCE/cell
Total cells
Total SCEs
3.08 _+ 0.24 a 6.02 -+ 0.35 ~'d
130 169
1161 2283
136 144
6.48 _+ 2.41 b'c'd 5.54 _+ 1.82 b'c'd
20 20
220 249
11.00 -+ 1.43 b'c'd 12.45 - 2.76 b'c'd
164 (240) 148 290
8.20 _+ 2.71 b'c'd (9.23 _+ 3.33) 5.92 _+ 1.67 b'~'d 11.64 _+ 2.92 b'c'd
29
328
11.31 -+ 3.66 b'c'd
Values in parentheses are the results of a repeat sample °Values are mean _+ SEM of six controls and six patients with RAEB bValues are mean --- SD ~Difference was tested by comparing the SCE of the cell population dp~O.O01 (Student's test), ep'~O.01.
No data No data
Mean SCE/cell 8.93 -+ 0.48 a 13.51 - 1.02 a'e
154
P.B. Murthy et al.
The two patients with RA in transformation exhibited SCE levels at 8.20 +_ 2.71 and 5.92 -+ 1.67 in BM. A repeat sample of the former patient revealed a rate of 9.23 _+ 3.33. SCE data on lymphocytes could be obtained from only one of the two patients and showed a near-normal level. BM from one patient with RA with ring sideroblasts exhibited the highest value among all the patients (11.64 _+ 2.92). The interchromosomal distribution of SCE in BM and l y m p h o c y t e s from controls and patients is presented in Tables 3 and 4. Analysis of observed and expected SCE in different chromosomes or chromosome groups revealed considerable nonrandomness, with an excess of SCEs in group B chromosomes and a deficiency in chromosomes of groups E, F, and G in both controls and patients. RAEB showed an u n e x p e c t e d excess of SCEs in group C as well. The data presented in Table 5 indicate that the SCE frequency in patients was not influenced by the BM karyotype status. DISCUSSION
In the present study, SCE frequency in BM cells of hematologically healthy subjects ranged between 2.41 and 3.76, with a mean of 3.08 -+ 0.24. Previous studies [4] have reported a slightly higher spontaneous level in normal BM, w h i c h ranged between 3.25 [14] and 6.00 [6]. One potential reason for this discrepancy could be the concentration of BrdU used in the culture [4] and the heterogeneous nature of BM cells [4, 6]. In the present study, we used a BrdU concentration of 3.07 p~g/m1-1. The results presented here clearly indicate a twofold increase in SCE frequency in BM cells of patients with RA, RAEB, and RAEB in transformation. The one patient with SA exhibited more than a threefold increase. In contrast, l y m p h o c y t e s from these patients showed only a marginal increase in SCE levels. Even though SCE rates in l y m p h o c y t e s from RAEB differed significantly from those of controls, we believe that this elevation is not striking. A few other reports have noted a similar increase in SCE in bone marrow cells of megaloblastic anemia [15] and refractory anemia [7]. Furthermore, Seshadri et al. [16], studying the l y m p h o c y t e s of patients with aplastic anemia (an acquired form of refractory anemia), have reported a significantly higher SCE. The interchromosomal distribution of SCE showed a few interesting observations. The n o n r a n d o m distribution of SCE, particularly with regard to groups B, E, F, and G, was also noted by others [15, 17]. A d d i t i o n a l l y , we found an excess of SCEs in group C chromosomes in both BM and l y m p h o c y t e s of RAEB. These data are in contrast to those reported by Carbone et al. [7], who found a preferential involvement of chromosome #1. The observation that group C chromosomes in RAEB were more c o m m o n l y involved in SCE formation should be regarded as important. It has been shown that most patients with RA and RAEB have n o n r a n d o m chromosomal changes involving chromosomes #7 and #8, in a d d i t i o n to 5 q - [8]. Because we did not use a c o m b i n e d G-banding and SCE method, it was not possible to discover w h i c h chromosome(s) was p r e d o m i n a n t l y involved for SCE in RAEB group C. The m e c h a n i s m by w h i c h SCEs are elevated in BM cells of MDS is not known. We have observed that most of these patients also present a defect in DNA repair [18], and it has been suggested that SCEs arise during DNA repair [17]. Unlike Knuutila et al. [15], we did not find a relation between SCE and karyotype status in the patients. It is possible that elevated SCE in our patients could be due to a toxic factor to which these patients were exposed prior to the d e v e l o p m e n t of BM abnormalities. In our sample, almost all patients were exposed to the atomic bomb in Hiroshima
155
g o
I N
g N NNdM
M ~ d M N ~ M ~
~
I N
© =
X N N o
N
g
a
gg P
o
156
o
o
E R
%
E
L) u3
0
..m
©
% 0
R _= >¢
>~%
gg
SCE i n M O S
Table 5
15 7
I n f l u e n c e of K a r y o t y p e S t a t u s o n SCE i n B M of P a t i e n t s Karyotypically normal cells
Abnormal cells
Patient
No.
SCEs
Mean ± SD
No.
SCEs
Mean ± SD
RAEB RA Case 1 Case 2 RAEB in transformation Case 1° Case 2 RA with ring sideroblasts
61
355
5.82 ± 1.66
87
536
6.16 ± 1.99 b
10 11
60 55
6.00 ± 1.95 5.00 ± 2.33
11 15
76 89
6.91 ± 2.55 ~ 5.93 ± 3.00 b
23 9 25
184 56 290
8.00 ± 3.73 6.22 ± 1.75 11.64 _~2.92
23 16
220 92
9.57 ± 2.83 b 5.75 ± 2.00 b
aPooled results from two experiments. bp-Not significantly different from karyotypically normal cells by Student's test. b e f o r e t h e y d e v e l o p e d t h e B M h y p e r p l a s i a . H o w e v e r , i n a n e a r l i e r s t u d y [19], w e d i d n o t find a n y a l t e r a t i o n i n SCE i n h e a l t h y a t o m i c b o m b s u r v i v o r s . T h u s , it app e a r s t h a t e l e v a t e d SCE i n p a t i e n t s w i t h M D S is a c h a r a c t e r i s t i c of t h e d i s e a s e . T h e f u n c t i o n a l s i g n i f i c a n c e of e l e v a t e d SCE i n M D S is d i f f i c u l t to d e t e r m i n e . A l t h o u g h t h e c e l l u l a r s i g n i f i c a n c e of SCE is far f r o m c l e a r [1, 2, 17], it h a s b e e n s u g g e s t e d t h a t SCE e l e v a t i o n is a c c o m p a n i e d b y a rise i n t h e m u t a t i o n f r e q u e n c y [20]. A s s t a t e d before, m a l i g n a n t t i s s u e s e x h i b i t e i t h e r s l i g h t l y i n c r e a s e d or r e d u c e d SCE r a t e s [4, 5]. It is n o t k n o w n w h y t h e s e p r e l e u k e m i c p a t i e n t s w i t h e l e v a t e d SCE t e n d to s h o w n o r m a l or r e d u c e d SCE w h e n t h e y d e v e l o p a c u t e l e u k e m i a s . This study is supported in part by a grant from the Ministry of Education. P.B.M. is a recipient of a fellowship from the Ministry of Education, Science and Culture of Japan.
REFERENCES 1. Latt SA (1981): Sister chromatid exchange formation. A n n u Rev Genet 15:11-15. 2. Wolff S, Morgan WF (1982): Modulating factors in sister chromatid exchange induction by mutagenic carcinogenes. In: Sister chromatid exchange, AA Sandberg, ed. Alan R. Liss, New York pp 515-533. 3. Chaganti RSK, Schonberg S, German J (1974): A manyfold increase in sister chromatid exchanges in Bloom's syndrome lymphocytes. Proc Natl Acad Sci USA 71:4508-4512. 4. Sandberg AA (1982): Sister chromatid exchange in h u m a n states. In: Sister chromatid exchange, AA Sandberg, ed. Alan R. Liss, New York, pp 619-651. 5. Sandberg AA (1979): Some comments on sister chromatid exchange (SCE) in h u m a n neoplasia. Cancer Genet Cytogenet 1:197-206. 6. Knuutila S, Helminen E, Vuopio P, de la Chapelle A (1978): Sister chromatid exchanges in h u m a n bone marrow cells. I. Control subjects and patients with leukemia. Hereditas 88:189-196. 7. Carbone P, Barbata G, Granata G, Margiotta G, Caronia F (1981): Sister chromatid exchange distribution in bone marrow cell chromosomes from patients with refractory anemia. Acta Haematol 65:177-182. 8. Nowell PC (1982): Cytogenetics of preleukemia. Cancer Genet Cytogenet 5:265-278. 9. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR, Sultan C (1982): Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 51:189-199.
158
P . B . M u r t h y et al.
10. Boyum A (1968): Separation of leukocytes from blood and bone marrow. Scand J Clin Lab Invest 21(Suppl):77-97. 11. Kamada N, Dohy H, Okada K, Oguma N, Kuramoto A, Tanaka K, Uchino H (1981): In vivo and in vitro activity of neutrophil alkaline phosphatase in acute myelocytic leukemia with 8;21 translocation. Blood 58:1213-1217. 12. Perry P, Wolff S (1974): New Giemsa method for the differential staining of sister chromatids. Nature 251:156-158. 13. ISCN (1978): An international system for h u m a n cytogenetic nomenclature. Cytogenet Cell Genet 21:309-404. 14. Shiraishi Y, Freeman AI, Sandberg AA (1976): Increased sister chromatid exchange in bone marrow and blood cells from Bloom's syndrome. Cytogenet Cell Genet 17:162-173. 15. Knuutila S (1982): Sister chromatid exchange in bone marrow cells. In: Sister chromatid exchange, AA Sandberg, ed. Alan R. Liss, New York, pp 165-173. 16. Seshadri R, Baker E, Leonard P, Sutherland G, Morley AA (1981): Sister chromatid exchange in aplastic anemia. Cancer Genet Cytogenet 3:81-84. 17. Wolff S (1977): Sister chromatid exchange. Ann Rev Genet 11:183-201. 18. Murthy PB, Kamada N, Kuramoto A (1984): Defective UV-induced DNA repair in bone marrow cells and peripheral lymphocytes of patients with refractory anemia with excess of blasts. Jpn J Clin Oncol 14:87-91. 19. Pant GS, Kamada N, Tanaka R (1976): Sister chromatid exchanges in peripheral lymphocytes of atomic bomb survivors and of normal individuals exposed to radiation and chemical agents. Hiroshima J Med Sci 25:99-105. 20. Carrano AV, Thompson LH, Lindl PA, Minkler JL (1978): Sister-chromatid exchange as an indicator of mutagenesis, Nature 271:551-553.
ABSTRACT: The frequency and distribution of sister chromatid exchange (SCE) was determined in bone marrow and peripheral lymphocytes of patients with preleukemic myelodysplastic syndromes. Patients with refractory anemia (RA), RA with excess of blasts (REAB), RA with ring sideroblasts, and RA in transformation presented a 2-3-fold increase in SCE frequency in the bone marrow cells. In contrast, lymphocytes from these patients showed only a marginal increase in SCE. Analysis of interchromosomal distribution of SCE indicated a preferential involvement of chromosomes in group C in patients with RA with excess of blasts. Furthermore, the SCE in patients was not found to be influenced by the karyotype status. INTRODUCTION Analysis of sister chromatid exchange (SCE) in b r o m o d e o x y u r i d i n e (BrdU) labeled chromosomes has been suggested to be a sensitive parameter of c h r o m o s o m a l instability [1,2]. Since the initial description of a manyfold increase in SCE frequency in cells from Bloom's s y n d r o m e [3], a search for a possible significance of SCE in various h u m a n states has been initiated. Studies on the a p p l i c a t i o n of SCE in h u m a n malignant and n e o p l a s i a - p r o n e situations have been a i m e d at looking into the probable diagnostic and prognostic importance of SCE in these diseases [4]. The results of these studies [4, 5], albeit controversial, have pointed to either slightly elevated or significantly r e d u c e d SCE in malignant tissues. P u b l i s h e d reports dealing with SCE in p r e l e u k e m i c m y e l o d ysplastic syndromes (MDS) have been few and have resulted in conflicting data [6, 7]. While Carbone et al. [7] have reported an elevated SCE frequency, particularly in chromosome #1, in cells from patients with refractory anemia (RA), Knuutila et al. [6] could not find such an alteration in patients with refractory i d i o p a t h i c sideroblastic anemia (SA). In view of this controversy, and in view of the fact that patients with MDS present an increased risk for acute leukemia [8], a study on the frequency of SCE in MDS becomes meaningful. We present the results of our studies on the frequency and distribution of SCE in bone marrow (BM) cells and peFrom the Department of Internal Medicine, Research Institute for Nuclear Medicine and Biology,Hiroshima University, Hiroshima, Japan. Address requests for reprints to Dr. Nanao Kamada, Department of Internal Medicine, Research Institute for Nuclear Medicine and Biology, Hiroshima University, 1-2-3 Kasumi-cho, Hirashima 734, Japan. Received December 29, 1983; accepted March 30, 1984.
151 © 1985 by Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017
Cancer Genetics and Cytogenetics15,151 158 (1985) 0165-4608/85/$03.30
15 2
P . B . Murthy et al.
ripheral l y m p h o c y t e s of patients with MDS. In addition, we attempt to analyze the frequency of SCE in relation to the BM karyotype status of the patients. MATERIALS AND METHODS
Eleven patients diagnosed as having MDS according to the French-American-British classification [9] were investigated. There were six with RA with excess of blasts (RAEB), two with RA, two with RAEB in transformation, and one with SA. Six patients with hematologically normal BM were also investigated. BM from patients prior to therapy and from controls was obtained, and mononuclear cells were separated by the Ficoll m e t h o d [10]. After Ficoll separation, the majority of the cell p o p u l a t i o n in most subjects from the two groups, was c o m p r i s e d of immature granulocytes, erythroblasts, lymphocytes, and monocytes in that order. A part of the m o n o n u c l e a r cells separated from the BM was used for direct karyotype investigation on G-banded preparations using a standard technique described previously [11], and the rest of it was used for SCE investigation, as described below. Two samples of BM cells of 3 x 105 cells each were cultured in RPMI 1640 m e d i u m s u p p l e m e n t e d with 10% fetal calf serum, L-glutamine, and antibiotics. BrdU (Sigma, St. Louis, MO) at a final concentration of 10p.M was a d d e d to the cells, and the cultures incubated at 37°C in the dark for 48 hr. Colchicine (0.05 ~g/ ml) was a d d e d during the last 1 hr of incubation. Cells were collected, hypotonically shocked (0.075 M KC1), fixed, and w a s h e d with methanol and acetic acid (3:1) and d r o p p e d onto cleaned slides. Air-dried preparations were exposed to Hoechst 33258 (0.5 ~g/ml) in p h o s p h a t e buffer (pH 7.0), exposed to ultraviolet light, and stained with 4% Giemsa [12]. Peripheral blood samples were also obtained from patients and controls at the time of collection of BM. Lymphocytes in buffy coat were cultured in RPMI 1640 m e d i u m with the same s u p p l e m e n t s as described above. Phytohemagglutinin (Wellcome) was a d d e d to cells and the cells incubated at 37°C for 24 hr. After this period, BrdU, at a final concentration of 10 p.M, was introduced into the cultures and the cells incubated for an additional 48 hr. After colchicine arrest, metaphase preparations were m a d e and stained for SCE as described above. SCEs were scored in at least 20 second-division metaphases from each BM and peripheral l y m p h o c y t e preparation. Agreement between two investigators was obtained before each SCE was scored. All metaphases scored were subsequently photographed, and karyotypes were prepared according to ISCN [13]. SCEs were identified in i n d i v i d u a l chromosomes or chromosome groups. RESULTS
Table 1 presents the clinical and hematologic data of the patients, as well as the results of the karyotype investigations. Data on the mean frequencies of SCEs are presented in Table 2. SCE frequencies in BM of six controls ranged between 2.41 and 3.76, with a mean of 3.08 ± 0.24 (mean ± SE). The corresponding mean values in patients with RAEB was 6.02 ± 0.35 (range 5.08-7.17). This difference was statistically significant. Lymphocytes from RAEB showed a mean SCE rate of 13.51 -+ 1.02, w h i c h differed significantly from that of 8.93 ± 0.48 seen in controls. The mean SCE frequencies in BM cells from two patients with RA were 6.48 ± 2.41 (mean ± SD) and 5.54 ± 1.82, each of w h i c h varied significantly with the mean level seen in control BM cells. Peripheral lymphocytes in RA showed only a marginal increase in SCE levels.
153 Table 1
C l i n i c a l , H e m a t o l o g i c , a n d K a r o t y p e D a t a of P a t i e n t s
Patients RAEB RAEB RAEB RAEB RAEB
1, 2, 3, 4, 5,
M, 69 M, 69 M, 68 F, 63 M, 54
RAEB 6, F, 74
WBC (>< 109/L)
NCC
Blasts + pro
6.0 7.2 5.7 1.6 3.7
8.7 8.6 9.5 7.1 8.5
178 42 121 150 99
Hyper Hyper Normo Hyper Normo
8.0 7.0 6.5 7.0 7.0
9.8
5.9
117
Normo
6.8
7.1 6.8
79 79
Hyper Hyper
5.7 5.1
8.1 8.7
21 135
Hyper Hyper
22.8 17.2
5.7
158
Hyper
4.0
RA 1, M, 79 2.1 RA 2, F, 82 1.9 RAEB in transformation Case 1, M, 47 1.3 Case 2, M, 78 4.1 RA w i t h ring sideroblasts Case 1, M, 66 3.5
Table 2
Hb PLts (g/dl) ( x 109/L)
Karyotype 46,XY, - 13, + 18/45,XY, - 13/46,XY 46,XY/47,XY, + 18 46,XY/45,XY,- 8 46,XX 45,XY, - 8,del(5)(q14),16q + ,17p + / 44,XY, - 8, - 9,del(5)(q14), 16q+,17p+ 47,XX,del(3)(q11),t(4;17) (p16;p13),del(5)(q13), del(12)(p12),t(12;?)(p13;?), del(15)(q22), + 17/ 46,XX,del(3)(q11), t(4;17)(p16;p13),del(5)(q13), del(12)(p12),t(12;?)(p13;?), del(15)(q22), + 17, - 20 46,XY, + 9, - 17/46,XY 46,XX
47,XY, + 8/46,XY 46,XY/46,XY,del(5)(q21) 46,XY
SCE F r e q u e n c y i n P a t i e n t s w i t h M D S BM
Subject Control RAEB RA Case 1 Case 2 RAEB in transformation Case 1 Case 2 RA w i t h ring sideroblasts
Total ceils
Total SCEs
171 148
527 891
21 26 20 (26) 25 25
Lymphocyte Mean SCE/cell
Total cells
Total SCEs
3.08 _+ 0.24 a 6.02 -+ 0.35 ~'d
130 169
1161 2283
136 144
6.48 _+ 2.41 b'c'd 5.54 _+ 1.82 b'c'd
20 20
220 249
11.00 -+ 1.43 b'c'd 12.45 - 2.76 b'c'd
164 (240) 148 290
8.20 _+ 2.71 b'c'd (9.23 _+ 3.33) 5.92 _+ 1.67 b'~'d 11.64 _+ 2.92 b'c'd
29
328
11.31 -+ 3.66 b'c'd
Values in parentheses are the results of a repeat sample °Values are mean _+ SEM of six controls and six patients with RAEB bValues are mean --- SD ~Difference was tested by comparing the SCE of the cell population dp~O.O01 (Student's test), ep'~O.01.
No data No data
Mean SCE/cell 8.93 -+ 0.48 a 13.51 - 1.02 a'e
154
P.B. Murthy et al.
The two patients with RA in transformation exhibited SCE levels at 8.20 +_ 2.71 and 5.92 -+ 1.67 in BM. A repeat sample of the former patient revealed a rate of 9.23 _+ 3.33. SCE data on lymphocytes could be obtained from only one of the two patients and showed a near-normal level. BM from one patient with RA with ring sideroblasts exhibited the highest value among all the patients (11.64 _+ 2.92). The interchromosomal distribution of SCE in BM and l y m p h o c y t e s from controls and patients is presented in Tables 3 and 4. Analysis of observed and expected SCE in different chromosomes or chromosome groups revealed considerable nonrandomness, with an excess of SCEs in group B chromosomes and a deficiency in chromosomes of groups E, F, and G in both controls and patients. RAEB showed an u n e x p e c t e d excess of SCEs in group C as well. The data presented in Table 5 indicate that the SCE frequency in patients was not influenced by the BM karyotype status. DISCUSSION
In the present study, SCE frequency in BM cells of hematologically healthy subjects ranged between 2.41 and 3.76, with a mean of 3.08 -+ 0.24. Previous studies [4] have reported a slightly higher spontaneous level in normal BM, w h i c h ranged between 3.25 [14] and 6.00 [6]. One potential reason for this discrepancy could be the concentration of BrdU used in the culture [4] and the heterogeneous nature of BM cells [4, 6]. In the present study, we used a BrdU concentration of 3.07 p~g/m1-1. The results presented here clearly indicate a twofold increase in SCE frequency in BM cells of patients with RA, RAEB, and RAEB in transformation. The one patient with SA exhibited more than a threefold increase. In contrast, l y m p h o c y t e s from these patients showed only a marginal increase in SCE levels. Even though SCE rates in l y m p h o c y t e s from RAEB differed significantly from those of controls, we believe that this elevation is not striking. A few other reports have noted a similar increase in SCE in bone marrow cells of megaloblastic anemia [15] and refractory anemia [7]. Furthermore, Seshadri et al. [16], studying the l y m p h o c y t e s of patients with aplastic anemia (an acquired form of refractory anemia), have reported a significantly higher SCE. The interchromosomal distribution of SCE showed a few interesting observations. The n o n r a n d o m distribution of SCE, particularly with regard to groups B, E, F, and G, was also noted by others [15, 17]. A d d i t i o n a l l y , we found an excess of SCEs in group C chromosomes in both BM and l y m p h o c y t e s of RAEB. These data are in contrast to those reported by Carbone et al. [7], who found a preferential involvement of chromosome #1. The observation that group C chromosomes in RAEB were more c o m m o n l y involved in SCE formation should be regarded as important. It has been shown that most patients with RA and RAEB have n o n r a n d o m chromosomal changes involving chromosomes #7 and #8, in a d d i t i o n to 5 q - [8]. Because we did not use a c o m b i n e d G-banding and SCE method, it was not possible to discover w h i c h chromosome(s) was p r e d o m i n a n t l y involved for SCE in RAEB group C. The m e c h a n i s m by w h i c h SCEs are elevated in BM cells of MDS is not known. We have observed that most of these patients also present a defect in DNA repair [18], and it has been suggested that SCEs arise during DNA repair [17]. Unlike Knuutila et al. [15], we did not find a relation between SCE and karyotype status in the patients. It is possible that elevated SCE in our patients could be due to a toxic factor to which these patients were exposed prior to the d e v e l o p m e n t of BM abnormalities. In our sample, almost all patients were exposed to the atomic bomb in Hiroshima
155
g o
I N
g N NNdM
M ~ d M N ~ M ~
~
I N
© =
X N N o
N
g
a
gg P
o
156
o
o
E R
%
E
L) u3
0
..m
©
% 0
R _= >¢
>~%
gg
SCE i n M O S
Table 5
15 7
I n f l u e n c e of K a r y o t y p e S t a t u s o n SCE i n B M of P a t i e n t s Karyotypically normal cells
Abnormal cells
Patient
No.
SCEs
Mean ± SD
No.
SCEs
Mean ± SD
RAEB RA Case 1 Case 2 RAEB in transformation Case 1° Case 2 RA with ring sideroblasts
61
355
5.82 ± 1.66
87
536
6.16 ± 1.99 b
10 11
60 55
6.00 ± 1.95 5.00 ± 2.33
11 15
76 89
6.91 ± 2.55 ~ 5.93 ± 3.00 b
23 9 25
184 56 290
8.00 ± 3.73 6.22 ± 1.75 11.64 _~2.92
23 16
220 92
9.57 ± 2.83 b 5.75 ± 2.00 b
aPooled results from two experiments. bp-Not significantly different from karyotypically normal cells by Student's test. b e f o r e t h e y d e v e l o p e d t h e B M h y p e r p l a s i a . H o w e v e r , i n a n e a r l i e r s t u d y [19], w e d i d n o t find a n y a l t e r a t i o n i n SCE i n h e a l t h y a t o m i c b o m b s u r v i v o r s . T h u s , it app e a r s t h a t e l e v a t e d SCE i n p a t i e n t s w i t h M D S is a c h a r a c t e r i s t i c of t h e d i s e a s e . T h e f u n c t i o n a l s i g n i f i c a n c e of e l e v a t e d SCE i n M D S is d i f f i c u l t to d e t e r m i n e . A l t h o u g h t h e c e l l u l a r s i g n i f i c a n c e of SCE is far f r o m c l e a r [1, 2, 17], it h a s b e e n s u g g e s t e d t h a t SCE e l e v a t i o n is a c c o m p a n i e d b y a rise i n t h e m u t a t i o n f r e q u e n c y [20]. A s s t a t e d before, m a l i g n a n t t i s s u e s e x h i b i t e i t h e r s l i g h t l y i n c r e a s e d or r e d u c e d SCE r a t e s [4, 5]. It is n o t k n o w n w h y t h e s e p r e l e u k e m i c p a t i e n t s w i t h e l e v a t e d SCE t e n d to s h o w n o r m a l or r e d u c e d SCE w h e n t h e y d e v e l o p a c u t e l e u k e m i a s . This study is supported in part by a grant from the Ministry of Education. P.B.M. is a recipient of a fellowship from the Ministry of Education, Science and Culture of Japan.
REFERENCES 1. Latt SA (1981): Sister chromatid exchange formation. A n n u Rev Genet 15:11-15. 2. Wolff S, Morgan WF (1982): Modulating factors in sister chromatid exchange induction by mutagenic carcinogenes. In: Sister chromatid exchange, AA Sandberg, ed. Alan R. Liss, New York pp 515-533. 3. Chaganti RSK, Schonberg S, German J (1974): A manyfold increase in sister chromatid exchanges in Bloom's syndrome lymphocytes. Proc Natl Acad Sci USA 71:4508-4512. 4. Sandberg AA (1982): Sister chromatid exchange in h u m a n states. In: Sister chromatid exchange, AA Sandberg, ed. Alan R. Liss, New York, pp 619-651. 5. Sandberg AA (1979): Some comments on sister chromatid exchange (SCE) in h u m a n neoplasia. Cancer Genet Cytogenet 1:197-206. 6. Knuutila S, Helminen E, Vuopio P, de la Chapelle A (1978): Sister chromatid exchanges in h u m a n bone marrow cells. I. Control subjects and patients with leukemia. Hereditas 88:189-196. 7. Carbone P, Barbata G, Granata G, Margiotta G, Caronia F (1981): Sister chromatid exchange distribution in bone marrow cell chromosomes from patients with refractory anemia. Acta Haematol 65:177-182. 8. Nowell PC (1982): Cytogenetics of preleukemia. Cancer Genet Cytogenet 5:265-278. 9. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR, Sultan C (1982): Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 51:189-199.
158
P . B . M u r t h y et al.
10. Boyum A (1968): Separation of leukocytes from blood and bone marrow. Scand J Clin Lab Invest 21(Suppl):77-97. 11. Kamada N, Dohy H, Okada K, Oguma N, Kuramoto A, Tanaka K, Uchino H (1981): In vivo and in vitro activity of neutrophil alkaline phosphatase in acute myelocytic leukemia with 8;21 translocation. Blood 58:1213-1217. 12. Perry P, Wolff S (1974): New Giemsa method for the differential staining of sister chromatids. Nature 251:156-158. 13. ISCN (1978): An international system for h u m a n cytogenetic nomenclature. Cytogenet Cell Genet 21:309-404. 14. Shiraishi Y, Freeman AI, Sandberg AA (1976): Increased sister chromatid exchange in bone marrow and blood cells from Bloom's syndrome. Cytogenet Cell Genet 17:162-173. 15. Knuutila S (1982): Sister chromatid exchange in bone marrow cells. In: Sister chromatid exchange, AA Sandberg, ed. Alan R. Liss, New York, pp 165-173. 16. Seshadri R, Baker E, Leonard P, Sutherland G, Morley AA (1981): Sister chromatid exchange in aplastic anemia. Cancer Genet Cytogenet 3:81-84. 17. Wolff S (1977): Sister chromatid exchange. Ann Rev Genet 11:183-201. 18. Murthy PB, Kamada N, Kuramoto A (1984): Defective UV-induced DNA repair in bone marrow cells and peripheral lymphocytes of patients with refractory anemia with excess of blasts. Jpn J Clin Oncol 14:87-91. 19. Pant GS, Kamada N, Tanaka R (1976): Sister chromatid exchanges in peripheral lymphocytes of atomic bomb survivors and of normal individuals exposed to radiation and chemical agents. Hiroshima J Med Sci 25:99-105. 20. Carrano AV, Thompson LH, Lindl PA, Minkler JL (1978): Sister-chromatid exchange as an indicator of mutagenesis, Nature 271:551-553.