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Effects of cell fusion and deoxynucleosides on sister-chromatid exchanges in B-lymphoblastoid cell lines from 5 Bloom syndrome patients
Mutation Research, 199 (1988) 75-83
75
Elsevier MTR 04574
Effects of cell fusion and deoxynucleosides on sister-chromatid exchanges in B-lymphoblastoid cell lines from 5 Bloom syndrome patients Yukimasa Shiraishi Laboratory of Human Cytogenetics, Department of Anatomy, Kochi Medical School, Nankoku-City 781-51, Kochi (Japan) (Received 7 July 1987) (Revision received 2 October 1987) (Accepted 14 October 1987)
Keywords: Cell fusion; Deoxynucleosides; Sister-chromatid exchanges; Lymphoblastoid B cell lines; Bloom syndrome patients.
Summary The effect of cell fusion and deoxynucleosides (deoxyadenosine, dA; deoxyguanosine, dG; deoxycytidine, dC; thymidine, T) on sister-chromatid exchanges (SCEs) in Bloom syndrome (BS) was studied in two types of BrdU (bromodeoxyuridine)-sensitive and BrdU-resistant B-lymphoblastoid cell lines (LCLs) with respect to cellular proliferation in BrdU-labeled culture conditions. Cell fusion between BrdU-sensitive and BrdU-resistant BS B-LCLs did not exhibit complementation, although when any of the BS B-LCLs (retaining high SCE character) labeled with BrdU were fused with non-labeled normal cells, the hybrid ceils had a normal level of SCE at the first mitosis after fusion. Deoxycytidine addition showed no effect on SCEs in normal cells but decreased SCEs in BS cells from the baseline level of 70 SCEs/cell to about 60 SCE/cell. Purine deoxyribonucleosides (dG and dA) caused a significant concentration-dependent increase in SCE frequency both in normal and BS cells. Although T caused a 2-fold increase in normal SCEs, it highly decreased BS SCE from 70 SCEs/cell to 35 SCEs/cell. FrdU did not greatly affect BS SCE in the presence of BrdU and T. These observations indicate strongly that BS cells may have a low thymidine pool compared with normal cells, which could account for a more efficient BrdU substitution in the DNA thus potentiating the template effect on SCE.
Bloom's syndrome (BS) is an autosomal recessive genetic disorder characterized by stunted growth, sensitivity to sunlight, immunodeficiency, and marked predisposition to cancer (German, 1969). Although cultured lymphocytes, fibroblasts, and marrow cells of patients with BS exhibit high frequencies of chromosome breaks and rearrange-
Correspondence: Dr. Yukimasa Shiraishi, Laboratory of Human Cytogenetics, Department of Anatomy, Kochi Medical School, Nankoku-City 781-51, Kochi (Japan).
ments, the most prominent cytogenetic characteristic is an increased frequency of sister-chromatid exchanges (SCEs) in cells labeled with bromodeoxyuridine (BrdU) for 2 cell cycles (Chaganti et al., 1974; Shiraishi et al., 1976, 1982). Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (LCLs) have been established from BS patients and some of these BS B-LCLs retain the original increased SCE character in 100% of the cells (Shiraishi et al., 1982, 1983). However, controversial results have been reported concerning cellular proliferation when cells are cultured in the
0027-5107/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
76 presence of BrdU among BS B-LCLs with high incidence of SCE. In our previous studies (Shiraishi et al., 1982, 1983), we have demonstrated, using single and twin SCE analysis in BrdU-labeled endomitosis, that most BS SCEs are caused by BrdU when BrdU-containing D N A is used as the template for replication and that BS B-LCLs are sensitive to BrdU with respect to rates of cellular proliferation. However, recently, Ray and German (1984) have pointed out that an EBV-transformed BS B-lymphoblastoid cell line with the characteristically elevated SCE frequency proliferates as does a normal B-LCL in BrdU-containing medium. More recently, we have also noticed that 2 EBVtransformed BS B-LCLs originating from 2 different BS patients exhibited BrdU resistance during cell proliferation in spite of similarly elevated frequencies of SCEs. Because of the fundamental importance to the understanding of BS of knowing whether there exist 2 complementation groups in BS, we have examined the effects of cell fusion on SCE between BrdU-sensitive and BrdU-resistant BS B-LCLs. Also, since a significant decrease in SCE rate in BS cells under co-culture conditions has been observed (Van Buul et al., 1978; Bartram et al., 1979; Rudiger et al., 1980; Bamezai and Shiraishi, 1986, 1987) and a possible role of low molecular weight metabolites ( < 1000 dalton) through gap-junctions was suggested, the effects of deoxyadenosine (dA), deoxycytidine (dC), deoxyguanosine (dG) and thymidine (T) in the medium on the frequency of SCE in BS (BrdU-sensitive and -resistant) and normal BLCLs were tested. Materials and methods
Permanent BS B-LCLs (BSI_2, BS2_2, BS-SYa, BS-SY4 and BS-SYs) and a normal B-LCL (KS86) were established from 5 patients with BS and from a normal subject by using EBV (Shiraishi et al., 1983a, b). As previously described, BSa_2 and BS2_2 cells were found to have the characteristically elevated frequency of SCE (average SCE value: 70.1 _+ 2.44 in BSI_2, 71.3 + 2.31 in BS2_2) in cells labeled with BrdU for 2 cell cycles. They also have chromosome instability and abnormal karyotypes: B81_2, 4 6 , X Y , 7 p + , 1 2 q + , + 14,t(15;15)(p11;q12)/ 46,XY,12q +,+14,t(15;15)(pll;q12); BS2.2, 47,
XY,2p+,t(3;15)(p25;q15),t(7;11)(q11;qlS), + m~/ 46,XY,2p +,t(3; 15)(pl 1 ;ql 5),t(7;11)(ql 1 ;pl 5). BSSY 1, BS-SY4 and BS-SY5 cell lines were newly established ones retaining high SCE character in 100% of cells (Table 1). The establishment of these 3 new cell lines was accomplished following the method previously described (Shiraishi et al., 1983a). Even though chromosome instabilities including breaks (6-8%) and quadriradials (0.01 0.02%) were detected at relatively low frequency, their karyotype was basically normal (46,XY in BS-SYa, 46,XY in BS-SY4 and 46,XY in BS-SYs), differing from those of BSI_2 and BS2.z. The karyotype of KS86 was normal, and the baseline level of SCEs/cell was 5.2 _+ 0.18. These cells were maintained in RPMI 1640 medium supplemented with 15% fetal calf serum (FCS) at 3 7 ° C in a 5% CO 2 humidified incubator. The protocol for cell fusion followed that described in a previous paper (Shiraishi et al., 1981). Briefly, BS B-lymphoblastoid cells were exposed to BrdU (10.0 ~ g / m l ) for 20 h (a period covering 1 round of D N A replication for most of the cells). Equal numbers (5 × 106) of labeled BS and unlabeled BS (or normal) cells were mixed, washed twice with RPMI 1640 medium and fused with commercial polyethylene glycol (50% PEG) (Boehringer Mannheim, Ltd.). 1 ml of 50% PEG was placed over the cell pellet and mixed well with a pipette for 1 min. 2 ml and then 4 ml of RPMI 1640 medium was added to PEG and mixed well for 2 and 4 min, respectively, each, and then 8 ml RPMI 1640 medium with 10% FCS was added. The cells were then centrifuged, and then reincubated without BrdU at 3 7 ° C in a CO 2 incubator and harvested every 24 h. The reincubation time after cell fusion was the same as in our previous study (Shiraishi et al., 1981). The incubation was continued for an additional 2 h in the presence of colcemid. Chromosome preparations and differential staining of sister chromatids were done with established techniques (Shiraishi et al., 1976). The effects of deoxynucleosides (dC, dA, dG, and T, 50, 100, and 200/zM) on the frequency of SCE were examined in the presence of BrdU at 10 # g / m l . Also, the effect of T with and without fluorodeoxyuridine (FrdU) (an inhibitor of thymidylate synthetase) on the SCE frequency was examined in the presence of BrdU at 10 btg/ml,
77
/cont
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,
i
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~
__c
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z
u 2
Z >_
BS1-2 c o n t
cont BUS BUIO BU20
~
i
BU5 BU~o O'-'~-'~t:3BU20 i
i
;
BS-SY5
• Days
i
BS-SYI
~
S
i
BS2-2 c o n t
rr 1
I
i
BUS B U~O oBU20
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Fig. 1. Cell growth curves of normal (KS86), and BS cells (BS1.2, BS2.2, BS-SY1, BS-SY4 and BS-SYs) in the presence and absence of BrdU (5, 10 and 20 # g / m l ) . As seen from this figure, BS-SY4 and BS-SY5 cells are resistant on cell growth, whereas BSI.2, BS2_2 and BS-SY1 cells are sensitive to BrdU.
78
since a low thymidine pool was suspected in BS cells. Results
BrdU effect on cell growth The cell growth curves of normal and BS BLCLs are shown in Fig. 1. In the control cultures, the growth patterns of these lines were very similar at the exponential growth phase, with their doubling times ranging from 30 to 36 h (Fig. 1), though BS cells grew slightly more slowly than the normal cells. The generation times (cell cycle time) of these lines were about 24 h. BrdU effects on normal and BS cell growth were studied by a cell viability test that used the trypan blue dye exclu-
sion method. Normal cells grew in the presence of various concentrations of BrdU (5, 10, and 20 /~g/ml). There were 2 types of BS B-LCLs, one is sensitive to BrdU and the other grew similarly to those of normal in the various BrdU concentrations. In BSa.2, BS2.2 and BS-SY1, very little cell growth was observed in BS cells in the presence of BrdU (5, 10, and 20 /~g/ml) after 2 days of culture, though BS-SY4 and BS-SY5 cells grew similarly to those of normal (KS86) cells in the presence of BrdU.
Effect of cell fusion on SCE Table 1 summarizes cytogenetic characterization of the B-lymphoblastoid cell lines established from a normal individual (KS86) and 5 patients
TABLE 1 F R E Q U E N C Y OF SISTER-CHROMATID E X C H A N G E S (SCEs) IN N O R M A L (KS86) A N D 5 BS B-LYMPHOBLASTOID CELL LINES LABELED W I T H BrdU FOR 2 CELL CYCLES Cell lines
Karyotype
Cells scored
SCEs/cell Range
Mean + S.E.
KS86
46,XX
100
2-11
5.6 _+0.33
BS~_2 BS2.2
46,XY,7p + ,12q + , + 14,t(15;15)(pl 1;q12) 47,XY,2p + ,t(3;15)(p25;q15),t(7;ll)(qll ;p15), + m I
85 100
50-97 51-96
69.9 + 2.81 70.4 + 2.42
BS-SY1 BS-SY4 BS-SY5
46,XY 46,XY 46,XX
80 75 100
48-97 54-95 59-91
68.6 + 2.16 72.1 + 2.33 71.6 +_2.61
TABLE 2 Q U A N T I T A T I V E EVALUATION OF SCE F R E Q U E N C I E S IN CELL F U S I O N BETWEEN KS86 CELLS A N D BrdU-LABELED BS B-LCLs Expt. No.
Cell line
First cell cycle
1
KS86 BS1. 2
BU
KS86 BS2.2
BU
KS86 BS-SY1
BU
KS86 BS-SY4
BU
KS86 BS-SY5
BU
2
3
4
5
Hybrid cells scored
SCEs/cell Range
Mean + S.E.
45
4-12
7.1 + 0.94
40
5-11
6.6+0.69
39
3-12
6.3+0.71
55
4-14
8.6+0.79
49
5-13
8.7+0.66
cell fusion
cell fusion
cell fusion
cell fusion
cell fusion
79
the hybrid cells were performed by a method described previously (Shiraishi et al., 1981). In the hybridization experiments, regardless of fusion combinations, no hybrid cells underwent mitosis until 48 h after fusion, indicating that their cell cycle time had been expanded beyond the normal range. After 24 h, all mitoses showed a diploid chromosome number, indicating that only unfused cells underwent mitosis in the first 24 h. On the basis of the mitotic index, taken every 24 h up to 72 h, the length of the cell cycle in hybrid
with BS. Table 2 presents the effects of cell fusion on the incidence of SCE. BS cells that had incorporated BrdU for one round of DNA replication (24 h) were fused with non-BrdU-labeled normal a n d / o r BS B-lymphoid cells. Thereafter, these cells were cultivated for 48 h in the absence of BrdU through another round of DNA replication. Thus, when the next mitoses were encountered, only the chromosomes of the cells which had incorporated BrdU had differentially labeled chromatids (Shiraishi et al., 1981). SCE observations in
i
$ t
Fig. 2. Hybrid metaphase of BrdU-labeled BS-SY4 and non-labeled KS86. Note that original BS SCEs (72.1_+2.33) are highly decreased to the level of about 11 SCE.
80
cells appeared to be similarly expanded. Hybrid cells obtained from the present fusion protocol usually contained tetraploid chromosome constitution and were detected at 10-15% of total mitotic cells. In Expts. 1-5, the protocol of cell fusion was the same as those described earlier (Shiraishi et al., 1981), and is reproduced for the purpose of comparing the effect of cell fusion on SCE frequency in BS cells. When BS cells labeled with BrdU were fused with non-treated normal cells, the hybrid cells had a normal level of SCE (approximately 6.3-8.7 SCEs/cell on the average) at the first mitosis after fusion. As shown in Table 2, both BrdU-sensitive and BrdU-resistant cells were similarly normalized by fusion with normal cells, though the SCE value of a hybrid between BrdUresistant and normal cells is slightly higher than that between BrdU-sensitive and normal cells.
Even though the SCE value of a hybrid between BrdU-labeled BS and non-labeled normal varied from 3 to 14 and was slightly higher than that of normal diploid cells, this increase is less than 2-fold that of normal cells. This variation of SCE in hybrid cells is possibly due to the difference in cell cycle timing at the time of cell fusion; it is not possible to normalize SCE which has occurred before the completion of cell fusion. Fig. 2 shows a hybrid metaphase of BrdU-labeled BS-SY4 and non-labeled KS86, revealing a highly decreased level of SCEs (about 11 SCEs) in a BS-SY4 cell. In Expts. 6-15, the SCE level of 10 cell fusion pairs between two BS B-LCLs of different origins was examined. As shown in Table 3, when BrdU-sensitive (or BrdU-resistant) cells labeled with BrdU (10/~g/ml) were fused with non-labeled BrdU-resistant (or BrdU-sensitive) cells, the hybrid cells still retained the original high SCE level and no
TABLE 3 Q U A N T I T A T I V E E V A L U A T I O N OF SCE F R E Q U E N C I E S IN H Y B R I D CELLS B E T W E E N D I F F E R E N T O R I G I N S O F BS PATIENTS Expt. No. 6
7
8
9
10
11
12
13
14
15
Cell line
First cell cycle
BS1. 2 BS2_2
BU
BSI. 2 BS-SY~
BU
BS1. 2 BS-SY4
BU
BSI. 2 BS-SY5
BU
BS2.2
H ybri d cells scored
SCEs/cell (mean 5: S.E.)
35
73.2 _ 3.01
29
69.9 +- 3.41
28
68.7+_2.87
25
70.1 +_3.01
31
76.4 + 2.94
39
66.6 + 2.49
28
65.9 + 2.09
25
67.4 +- 2.47
22
68.6 5:3.11
23
71.1 5:2.32
cell fusion
cell fusion
cell fusion
cell fusion
cell fusion
-
BS-S¥~
BU
BS2.2 BS-SY4
BU
cell fusion
BS2.2 BS-SY5
BU
cell fusion
BS-SY1 BS-SY4
BU
cell fusion
BS-SY 1
-
cell fusion
BS-SV,
BU
BS-SV4
-
BS-SY5
BU
cell fusion
81 El
$
I
•
•
",re. sj. .j ' ,"r,'-; I ' "
,, ,
IIt
Fig. 3. Hybrid metaphase of BrdU-labeled BS2.2 and non-labeled BS-SY4.
TABLE 4 EFFECTS OF NUCLEOSIDES, DEOXYCYTIDINE (dC), DEOXYGUANOSINE (dG), DEOXYADENOSINE (dA) AND THYMIDINE (T) ON SCE IN NORMAL (KS86), BS2_2 (BrdU-SENSITIVE) AND BS-SY4 (BrdU-RESISTANT) CELLS Expts.
BS B-lymphoblastoid cell lines
No.
Cells counted
KS86
BS2.2 SCEs + S.E.
BS-SY4
Cells counted
SCEs + S.E.
Cells counted
SCEs 5- S.E.
1
BU(10) a
80
5.1+0.04
60
70.4-t-2.42
50
72.1+2.33
2 3
BU(10) + dC 100/~M BU(10)+dC 200 #M
80 80
5.4+0.03 5.6-1-0.02
60 60
58.2+2.21 56.9+2.09
50 50
59.4+2.61 58.1 +2.29
4 5
BU(10)+dG 100 #M BU(10)+dG 200/~M
80 80
5.9+0.04 6.4+0.05
60 60
87.95-2.34 97.55-2.31
50 50
86.45-2.13 89.15-2.46
6 7
BU(10)+dA 100 #M BU(10)+ dA 200 #M
80 80
6.7+0.04 7.4+0.05
60 60
83.45-2.14 94.45-2.11
50 50
84.65-2.21 95.85-2.68
8 9
BU(10) + T 100/~ M BU(10) + T 200/xM
80 80
10.1 + 0.07 12.15-0.06
60 60
35.3 + 2.71 39.1+2.14
50 40
37.4 5-1.98 41.1+2.49
BU, BrdU; 10/~g/ml.
82 TABLE 5 EFFECTS OF THYMIDINE (T) AND FLUORODEOXYURIDINE (FU) ON SCE IN NORMAL (KS86), BS2.2 AND BS-SY4 CELLS Expts.
KS86 Cells counted
BS2.2 SCEs + S.E.
BS.SYa
Cells counted
SCEs + S.E.
Cells counted
SCEs 5: S.E.
BU(10) a
80
5.1 + 0.04
60
70.6 + 2.09
60
69.9 + 2.01
BU(10) + FU(0.1/*g/ml) BU(10) + FU(0.4 ~ g/ml)
40 40
9.9+0.05 11.4+0.06
40 40
71.8+2.48 71.7+2.37
40 40
70.65:2.41 71.1_+2.51
BU(10) + T(100/xM) BU(10) + T(200/xM)
80 80
10.1+0.07 12.1+0.06
60 60
35.3+2.71 39.1_+2.14
50 40
37.4+1.98 41.15:2.49
BU(10) + T(100/,M) + FU(0.1/~ g/ml) BU(10) + T(100 ~M) + FU(0.4/~ g/ml) BU(10) + T(200/~M) + FU(0.1 btg/ml) BU(10) + T(200/tM) + FU(0.4/~ g/ml)
40 40 40 40
12.2 + 0.06 13.75:0.07 13.75:0.05 14.15:0.08
40 40 35 35
35.9 5:2.49 36.1 5:2.66 38.45:2.33 39.85:2.45
40. 40 40 40
38.2 5:2.42 38.1 5:2.39 39.45:2.55 42.15:2.39
a BU, BrdU; 10/~g/ml.
pair of complementation was observed in the present experiment. Fig. 3 shows the hybrid metaphase of BrdU-labeled BS2.2 and non-labeled BSSY4, showing the original increased level of SCE in a BS2.2 cell. This strongly suggests that there may be no complementation group in BS.
Effects of nucleosides and FrdU on BS SCE The effect of dC, dG, dA and T on normal and BS SCEs was measured using KS86, BS1.2 (BrdUsensitive) and BS-SY4 (BrdU-resistant) (Table 4). In normal cells, T increased SCEs from 5.1 baseline level up to 12.1 SCEs/cell (average); dA and d G caused a small increase in SCEs, whereas dC showed no effect (Table 4). However, in BS cells (BSa_z, BS-SY4) T produced a significant reduction in SCE from a baseline of 70 to 35 SCEs/cell (Table 4). Table 5 shows the effect of FrdU, in the presence and absence of T, on SCE in normal (KS86) and BS (BS2_2, BS-SY1) cells. FrdU significantly increased SCE in normal cells but produced no noticeable effect on BS SCEs. T plus FrdU produced a slightly higher SCE level in normal cells but made no difference in BS SCE compared to T alone. This strongly suggests that BS has a T pool deficiency. Deoxycytidine caused an SCE reduction from approximately 70 to approximately 60 SCEs/cell. However, both purine deoxyribonucleosides, dA and dG, caused signifi-
cant increase of SCE in BS2. 2 and BS-SY4 cells (Table 4). Discussion Our study of EBV-transformed BS B-LCLs, in which every cell exhibits the characteristically elevated SCE frequency, shows that (1) there are two types (BrdU-sensitive and BrdU-resistant) of cellular proliferation in the BrdU-labeled culture condition; BS-SY4, 5 cells proliferate like normal cells when BrdU is added to their medium, and (2) cell fusion between BrdU-sensitive and BrdU-resistant BS B-LCLs exhibited no complementation, retaining original increased SCE levels, whereas when any of the BS cells labeled with BrdU were fused with non-labeled normal cells, the hybrid cells had almost a normal level of SCE at the first mitosis after fusion. The data presented confirm our earlier observation (Shiraishi et al., 1981) that fusion of BrdU-labeled BS cells with non-labeled normal cells results in correction of the SCE level, indicating that BS cells may have a missing or defective section of D N A which is readily complemented by cell fusion. The origin and the mechanism of high SCE frequency in BS cells grown in BrdU, as compared with normal cells, are obscure. This study is also an attempt to test the contributions of the de-
83
oxynucleosides on the excess SCEs in BS cells, since DNA nucleoside pool bias is a reasonable interpretation of the co-cultivation experiments (Bamezai and Shiraishi, 1986). Addition of individual nucleosides (dA, dC, dG and T) to the growth medium caused significantly similar effects on the SCE frequency in all BS B-LCLs examined. Deoxycytidine addition showed no effect on SCEs in normal cells but decreased SCEs in BS cells from the baseline level of 70 SCEs/cell to about 60 SCEs/cell. Deoxycytidine is known to counteract the increase in SCEs by excess thymidine and BrdU in normal cells (Davidson et al., 1980; Kaufman, 1986). Purine deoxyribonucleosides (dG and dA) caused a significant concentration-dependent increase in SCE frequency both in normal and BS cells (Table 4). Although dT caused a 2-fold increase in normal SCEs, BS SCEs were inhibited about 50%, in both BrdU-sensitive and -resistant BS cells. Addition of FrdU did not greatly affect BS SCEs in the presence of BrdU alone and BrdU plus T. These observations suggest that BS cells with high SCE character may have a low thymidine pool compared with normal cells, which could also account for the spontaneous mutator effect of BS cells and perhaps also a more efficient BrdU substitution in DNA thus potentiating the template effect on SCEs. If this were the case, then the co-cultivation effect (see above) could be due to the transfer of a thymidine metabolite from normal into BS cells through gap-junctions. Direct pool measurements are necessary to test this hypothesis (experiments in progress) which could possibly lead to the elucidation of the molecular defect in BS cells.
Acknowledgements This work was supported in part by a grant-inaid from the Ministry of Education, Science and Culture of Japan, and from the Nissan Science Foundation. The author is grateful to Miss Michiyo Ozawa for her technical assistance.
References Bamezai, R., and Y. Shiraishi (1986) Cell cycle progression and SCE rate of Bloom syndrome cells with/without co-cultivation in the presence/absence of normal cells, Exp. Cell Res., 164, 163-173. Bamezai, R., and Y. Shiraishi (1987) Cell cycle rate and sister
chromatid exchange profile in polyethylene glycol-exposed/unexposed Bloom syndrome and normal cells, Hum. Genet., 75, 356-358. Bartram, C.R., H.W. Rudiger and E. Passarge (1979) Frequency of sister chromatid exchanges in Bloom syndrome fibroblasts reduced by co-cultivation with normal cells, Hum. Genet., 46, 331-334. Chaganti, R.S.K., S. Schonberg and J. German (1974) A manyfold increase in sister chromatid exchanges in Bloom's syndrome lymphocytes, Proc. Natl. Acad. Sci. (U.S.A.), 71, 4508-4512. Davidson, R.L., E.R. Kaufman, C.P. Dougherty, A.M. Ouellette, C.M. Difolco and S.A. Latt (1980) Induction of sister chromatid exchanges by BUdR is largely independent of the BUdR content of DNA, Nature (London), 284, 75-76. German, J. (1969) Bloom's syndrome, I. Genetical and clinical observations in the first twenty-seven patients, Am. J. Hum. Genet., 21, 196-227. Kaufman, E.R. (1986) Induction of sister chromatid exchanges by the replication of 5-bromouracil-substituted DNA under conditions of nucleotide-pool imbalance, Mutation Res., 163, 41-50. Ray, J.H., and J. German (1984) Bloom's syndrome and EM9 cells in BrdU-contalning medium exhibit similarly elevated frequencies of sister chromatid exchange but dissimilar amounts of cellular proliferation and chromosome disruption, Chromosoma, 90, 383-388. Rudiger, H.W., C.R. Bartram, W. Harder and E. Passarge (1980) Rate of sister chromatid exchanges in Bloom syndrome fibroblasts reduced by co-cultivation with normal fibroblasts, Am. J. Hum. Genet., 32, 150-157. Shiraishi, Y., A.F. Freeman and A.A. Sandberg (1976) Increased sister chromatid exchange in bone marrow and blood cells from Bloom's syndrome, Cytogenet. Cell Genet., 17, 162-173. Shiraishi, Y., S. Matsui and A.A. Sandberg (1981) Normalization by cell fusion of sister chromatid exchange in Bloom syndrome lymphocytes, Science, 212, 820-822. Shiraishi, Y., T.H. Yosida and A.A. Sandberg (1982) Analysis of single and twin sister chromatid exchanges in endoreduplicated normal and Bloom syndrome B-lymphoid cells, Chromosoma, 87, 1-8. Shiraishi, Y., I. Kubonishi and A.A. Sandberg (1983a) Establishment of B-lymphoid cell lines retaining cytogenetic characteristics of Bloom syndrome, Cancer Genet. Cytogenet., 56, 129-138. Shiraishi, Y., T.H. Yosida and A.A. Sandberg (1983b) Analyses of bromodeoxyuridine-associated sister chromatid exchanges (SCEs) in Bloom syndrome based on cell fusion: Single and twin SCEs in endoreduplication, Proc. Natl. Acad. Sci. (U.S.A.), 80, 4369-4373. Shiraishi, Y., S. Yosimoto, I. Miyoshi, N. Kondo, T. Orii and A.A. Sandberg (1983c) Dimorphism of sister chromatid exchange in Bloom syndrome B- and T-cell lines trans: formed with Epstein-Barr and adult T-cell leukemia viruses, Cancer Res., 43, 3836-3840. Van Buul, P.P.W., A.T. Natarajan and E.A.M. Verdegaal-Immerzeel (1978) Suppression of the frequencies of sister chromatid exchanges in Bloom's syndrome fibroblasts by co-cultivation with Chinese hamster cells, Hum. Genet., 44, 187-189.
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Elsevier MTR 04574
Effects of cell fusion and deoxynucleosides on sister-chromatid exchanges in B-lymphoblastoid cell lines from 5 Bloom syndrome patients Yukimasa Shiraishi Laboratory of Human Cytogenetics, Department of Anatomy, Kochi Medical School, Nankoku-City 781-51, Kochi (Japan) (Received 7 July 1987) (Revision received 2 October 1987) (Accepted 14 October 1987)
Keywords: Cell fusion; Deoxynucleosides; Sister-chromatid exchanges; Lymphoblastoid B cell lines; Bloom syndrome patients.
Summary The effect of cell fusion and deoxynucleosides (deoxyadenosine, dA; deoxyguanosine, dG; deoxycytidine, dC; thymidine, T) on sister-chromatid exchanges (SCEs) in Bloom syndrome (BS) was studied in two types of BrdU (bromodeoxyuridine)-sensitive and BrdU-resistant B-lymphoblastoid cell lines (LCLs) with respect to cellular proliferation in BrdU-labeled culture conditions. Cell fusion between BrdU-sensitive and BrdU-resistant BS B-LCLs did not exhibit complementation, although when any of the BS B-LCLs (retaining high SCE character) labeled with BrdU were fused with non-labeled normal cells, the hybrid ceils had a normal level of SCE at the first mitosis after fusion. Deoxycytidine addition showed no effect on SCEs in normal cells but decreased SCEs in BS cells from the baseline level of 70 SCEs/cell to about 60 SCE/cell. Purine deoxyribonucleosides (dG and dA) caused a significant concentration-dependent increase in SCE frequency both in normal and BS cells. Although T caused a 2-fold increase in normal SCEs, it highly decreased BS SCE from 70 SCEs/cell to 35 SCEs/cell. FrdU did not greatly affect BS SCE in the presence of BrdU and T. These observations indicate strongly that BS cells may have a low thymidine pool compared with normal cells, which could account for a more efficient BrdU substitution in the DNA thus potentiating the template effect on SCE.
Bloom's syndrome (BS) is an autosomal recessive genetic disorder characterized by stunted growth, sensitivity to sunlight, immunodeficiency, and marked predisposition to cancer (German, 1969). Although cultured lymphocytes, fibroblasts, and marrow cells of patients with BS exhibit high frequencies of chromosome breaks and rearrange-
Correspondence: Dr. Yukimasa Shiraishi, Laboratory of Human Cytogenetics, Department of Anatomy, Kochi Medical School, Nankoku-City 781-51, Kochi (Japan).
ments, the most prominent cytogenetic characteristic is an increased frequency of sister-chromatid exchanges (SCEs) in cells labeled with bromodeoxyuridine (BrdU) for 2 cell cycles (Chaganti et al., 1974; Shiraishi et al., 1976, 1982). Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (LCLs) have been established from BS patients and some of these BS B-LCLs retain the original increased SCE character in 100% of the cells (Shiraishi et al., 1982, 1983). However, controversial results have been reported concerning cellular proliferation when cells are cultured in the
0027-5107/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
76 presence of BrdU among BS B-LCLs with high incidence of SCE. In our previous studies (Shiraishi et al., 1982, 1983), we have demonstrated, using single and twin SCE analysis in BrdU-labeled endomitosis, that most BS SCEs are caused by BrdU when BrdU-containing D N A is used as the template for replication and that BS B-LCLs are sensitive to BrdU with respect to rates of cellular proliferation. However, recently, Ray and German (1984) have pointed out that an EBV-transformed BS B-lymphoblastoid cell line with the characteristically elevated SCE frequency proliferates as does a normal B-LCL in BrdU-containing medium. More recently, we have also noticed that 2 EBVtransformed BS B-LCLs originating from 2 different BS patients exhibited BrdU resistance during cell proliferation in spite of similarly elevated frequencies of SCEs. Because of the fundamental importance to the understanding of BS of knowing whether there exist 2 complementation groups in BS, we have examined the effects of cell fusion on SCE between BrdU-sensitive and BrdU-resistant BS B-LCLs. Also, since a significant decrease in SCE rate in BS cells under co-culture conditions has been observed (Van Buul et al., 1978; Bartram et al., 1979; Rudiger et al., 1980; Bamezai and Shiraishi, 1986, 1987) and a possible role of low molecular weight metabolites ( < 1000 dalton) through gap-junctions was suggested, the effects of deoxyadenosine (dA), deoxycytidine (dC), deoxyguanosine (dG) and thymidine (T) in the medium on the frequency of SCE in BS (BrdU-sensitive and -resistant) and normal BLCLs were tested. Materials and methods
Permanent BS B-LCLs (BSI_2, BS2_2, BS-SYa, BS-SY4 and BS-SYs) and a normal B-LCL (KS86) were established from 5 patients with BS and from a normal subject by using EBV (Shiraishi et al., 1983a, b). As previously described, BSa_2 and BS2_2 cells were found to have the characteristically elevated frequency of SCE (average SCE value: 70.1 _+ 2.44 in BSI_2, 71.3 + 2.31 in BS2_2) in cells labeled with BrdU for 2 cell cycles. They also have chromosome instability and abnormal karyotypes: B81_2, 4 6 , X Y , 7 p + , 1 2 q + , + 14,t(15;15)(p11;q12)/ 46,XY,12q +,+14,t(15;15)(pll;q12); BS2.2, 47,
XY,2p+,t(3;15)(p25;q15),t(7;11)(q11;qlS), + m~/ 46,XY,2p +,t(3; 15)(pl 1 ;ql 5),t(7;11)(ql 1 ;pl 5). BSSY 1, BS-SY4 and BS-SY5 cell lines were newly established ones retaining high SCE character in 100% of cells (Table 1). The establishment of these 3 new cell lines was accomplished following the method previously described (Shiraishi et al., 1983a). Even though chromosome instabilities including breaks (6-8%) and quadriradials (0.01 0.02%) were detected at relatively low frequency, their karyotype was basically normal (46,XY in BS-SYa, 46,XY in BS-SY4 and 46,XY in BS-SYs), differing from those of BSI_2 and BS2.z. The karyotype of KS86 was normal, and the baseline level of SCEs/cell was 5.2 _+ 0.18. These cells were maintained in RPMI 1640 medium supplemented with 15% fetal calf serum (FCS) at 3 7 ° C in a 5% CO 2 humidified incubator. The protocol for cell fusion followed that described in a previous paper (Shiraishi et al., 1981). Briefly, BS B-lymphoblastoid cells were exposed to BrdU (10.0 ~ g / m l ) for 20 h (a period covering 1 round of D N A replication for most of the cells). Equal numbers (5 × 106) of labeled BS and unlabeled BS (or normal) cells were mixed, washed twice with RPMI 1640 medium and fused with commercial polyethylene glycol (50% PEG) (Boehringer Mannheim, Ltd.). 1 ml of 50% PEG was placed over the cell pellet and mixed well with a pipette for 1 min. 2 ml and then 4 ml of RPMI 1640 medium was added to PEG and mixed well for 2 and 4 min, respectively, each, and then 8 ml RPMI 1640 medium with 10% FCS was added. The cells were then centrifuged, and then reincubated without BrdU at 3 7 ° C in a CO 2 incubator and harvested every 24 h. The reincubation time after cell fusion was the same as in our previous study (Shiraishi et al., 1981). The incubation was continued for an additional 2 h in the presence of colcemid. Chromosome preparations and differential staining of sister chromatids were done with established techniques (Shiraishi et al., 1976). The effects of deoxynucleosides (dC, dA, dG, and T, 50, 100, and 200/zM) on the frequency of SCE were examined in the presence of BrdU at 10 # g / m l . Also, the effect of T with and without fluorodeoxyuridine (FrdU) (an inhibitor of thymidylate synthetase) on the SCE frequency was examined in the presence of BrdU at 10 btg/ml,
77
/cont
K586
~
BU20 BUIO
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,
i
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BS-SYz,
~
__c
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z
u 2
Z >_
BS1-2 c o n t
cont BUS BUIO BU20
~
i
BU5 BU~o O'-'~-'~t:3BU20 i
i
;
BS-SY5
• Days
i
BS-SYI
~
S
i
BS2-2 c o n t
rr 1
I
i
BUS B U~O oBU20
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Fig. 1. Cell growth curves of normal (KS86), and BS cells (BS1.2, BS2.2, BS-SY1, BS-SY4 and BS-SYs) in the presence and absence of BrdU (5, 10 and 20 # g / m l ) . As seen from this figure, BS-SY4 and BS-SY5 cells are resistant on cell growth, whereas BSI.2, BS2_2 and BS-SY1 cells are sensitive to BrdU.
78
since a low thymidine pool was suspected in BS cells. Results
BrdU effect on cell growth The cell growth curves of normal and BS BLCLs are shown in Fig. 1. In the control cultures, the growth patterns of these lines were very similar at the exponential growth phase, with their doubling times ranging from 30 to 36 h (Fig. 1), though BS cells grew slightly more slowly than the normal cells. The generation times (cell cycle time) of these lines were about 24 h. BrdU effects on normal and BS cell growth were studied by a cell viability test that used the trypan blue dye exclu-
sion method. Normal cells grew in the presence of various concentrations of BrdU (5, 10, and 20 /~g/ml). There were 2 types of BS B-LCLs, one is sensitive to BrdU and the other grew similarly to those of normal in the various BrdU concentrations. In BSa.2, BS2.2 and BS-SY1, very little cell growth was observed in BS cells in the presence of BrdU (5, 10, and 20 /~g/ml) after 2 days of culture, though BS-SY4 and BS-SY5 cells grew similarly to those of normal (KS86) cells in the presence of BrdU.
Effect of cell fusion on SCE Table 1 summarizes cytogenetic characterization of the B-lymphoblastoid cell lines established from a normal individual (KS86) and 5 patients
TABLE 1 F R E Q U E N C Y OF SISTER-CHROMATID E X C H A N G E S (SCEs) IN N O R M A L (KS86) A N D 5 BS B-LYMPHOBLASTOID CELL LINES LABELED W I T H BrdU FOR 2 CELL CYCLES Cell lines
Karyotype
Cells scored
SCEs/cell Range
Mean + S.E.
KS86
46,XX
100
2-11
5.6 _+0.33
BS~_2 BS2.2
46,XY,7p + ,12q + , + 14,t(15;15)(pl 1;q12) 47,XY,2p + ,t(3;15)(p25;q15),t(7;ll)(qll ;p15), + m I
85 100
50-97 51-96
69.9 + 2.81 70.4 + 2.42
BS-SY1 BS-SY4 BS-SY5
46,XY 46,XY 46,XX
80 75 100
48-97 54-95 59-91
68.6 + 2.16 72.1 + 2.33 71.6 +_2.61
TABLE 2 Q U A N T I T A T I V E EVALUATION OF SCE F R E Q U E N C I E S IN CELL F U S I O N BETWEEN KS86 CELLS A N D BrdU-LABELED BS B-LCLs Expt. No.
Cell line
First cell cycle
1
KS86 BS1. 2
BU
KS86 BS2.2
BU
KS86 BS-SY1
BU
KS86 BS-SY4
BU
KS86 BS-SY5
BU
2
3
4
5
Hybrid cells scored
SCEs/cell Range
Mean + S.E.
45
4-12
7.1 + 0.94
40
5-11
6.6+0.69
39
3-12
6.3+0.71
55
4-14
8.6+0.79
49
5-13
8.7+0.66
cell fusion
cell fusion
cell fusion
cell fusion
cell fusion
79
the hybrid cells were performed by a method described previously (Shiraishi et al., 1981). In the hybridization experiments, regardless of fusion combinations, no hybrid cells underwent mitosis until 48 h after fusion, indicating that their cell cycle time had been expanded beyond the normal range. After 24 h, all mitoses showed a diploid chromosome number, indicating that only unfused cells underwent mitosis in the first 24 h. On the basis of the mitotic index, taken every 24 h up to 72 h, the length of the cell cycle in hybrid
with BS. Table 2 presents the effects of cell fusion on the incidence of SCE. BS cells that had incorporated BrdU for one round of DNA replication (24 h) were fused with non-BrdU-labeled normal a n d / o r BS B-lymphoid cells. Thereafter, these cells were cultivated for 48 h in the absence of BrdU through another round of DNA replication. Thus, when the next mitoses were encountered, only the chromosomes of the cells which had incorporated BrdU had differentially labeled chromatids (Shiraishi et al., 1981). SCE observations in
i
$ t
Fig. 2. Hybrid metaphase of BrdU-labeled BS-SY4 and non-labeled KS86. Note that original BS SCEs (72.1_+2.33) are highly decreased to the level of about 11 SCE.
80
cells appeared to be similarly expanded. Hybrid cells obtained from the present fusion protocol usually contained tetraploid chromosome constitution and were detected at 10-15% of total mitotic cells. In Expts. 1-5, the protocol of cell fusion was the same as those described earlier (Shiraishi et al., 1981), and is reproduced for the purpose of comparing the effect of cell fusion on SCE frequency in BS cells. When BS cells labeled with BrdU were fused with non-treated normal cells, the hybrid cells had a normal level of SCE (approximately 6.3-8.7 SCEs/cell on the average) at the first mitosis after fusion. As shown in Table 2, both BrdU-sensitive and BrdU-resistant cells were similarly normalized by fusion with normal cells, though the SCE value of a hybrid between BrdUresistant and normal cells is slightly higher than that between BrdU-sensitive and normal cells.
Even though the SCE value of a hybrid between BrdU-labeled BS and non-labeled normal varied from 3 to 14 and was slightly higher than that of normal diploid cells, this increase is less than 2-fold that of normal cells. This variation of SCE in hybrid cells is possibly due to the difference in cell cycle timing at the time of cell fusion; it is not possible to normalize SCE which has occurred before the completion of cell fusion. Fig. 2 shows a hybrid metaphase of BrdU-labeled BS-SY4 and non-labeled KS86, revealing a highly decreased level of SCEs (about 11 SCEs) in a BS-SY4 cell. In Expts. 6-15, the SCE level of 10 cell fusion pairs between two BS B-LCLs of different origins was examined. As shown in Table 3, when BrdU-sensitive (or BrdU-resistant) cells labeled with BrdU (10/~g/ml) were fused with non-labeled BrdU-resistant (or BrdU-sensitive) cells, the hybrid cells still retained the original high SCE level and no
TABLE 3 Q U A N T I T A T I V E E V A L U A T I O N OF SCE F R E Q U E N C I E S IN H Y B R I D CELLS B E T W E E N D I F F E R E N T O R I G I N S O F BS PATIENTS Expt. No. 6
7
8
9
10
11
12
13
14
15
Cell line
First cell cycle
BS1. 2 BS2_2
BU
BSI. 2 BS-SY~
BU
BS1. 2 BS-SY4
BU
BSI. 2 BS-SY5
BU
BS2.2
H ybri d cells scored
SCEs/cell (mean 5: S.E.)
35
73.2 _ 3.01
29
69.9 +- 3.41
28
68.7+_2.87
25
70.1 +_3.01
31
76.4 + 2.94
39
66.6 + 2.49
28
65.9 + 2.09
25
67.4 +- 2.47
22
68.6 5:3.11
23
71.1 5:2.32
cell fusion
cell fusion
cell fusion
cell fusion
cell fusion
-
BS-S¥~
BU
BS2.2 BS-SY4
BU
cell fusion
BS2.2 BS-SY5
BU
cell fusion
BS-SY1 BS-SY4
BU
cell fusion
BS-SY 1
-
cell fusion
BS-SV,
BU
BS-SV4
-
BS-SY5
BU
cell fusion
81 El
$
I
•
•
",re. sj. .j ' ,"r,'-; I ' "
,, ,
IIt
Fig. 3. Hybrid metaphase of BrdU-labeled BS2.2 and non-labeled BS-SY4.
TABLE 4 EFFECTS OF NUCLEOSIDES, DEOXYCYTIDINE (dC), DEOXYGUANOSINE (dG), DEOXYADENOSINE (dA) AND THYMIDINE (T) ON SCE IN NORMAL (KS86), BS2_2 (BrdU-SENSITIVE) AND BS-SY4 (BrdU-RESISTANT) CELLS Expts.
BS B-lymphoblastoid cell lines
No.
Cells counted
KS86
BS2.2 SCEs + S.E.
BS-SY4
Cells counted
SCEs + S.E.
Cells counted
SCEs 5- S.E.
1
BU(10) a
80
5.1+0.04
60
70.4-t-2.42
50
72.1+2.33
2 3
BU(10) + dC 100/~M BU(10)+dC 200 #M
80 80
5.4+0.03 5.6-1-0.02
60 60
58.2+2.21 56.9+2.09
50 50
59.4+2.61 58.1 +2.29
4 5
BU(10)+dG 100 #M BU(10)+dG 200/~M
80 80
5.9+0.04 6.4+0.05
60 60
87.95-2.34 97.55-2.31
50 50
86.45-2.13 89.15-2.46
6 7
BU(10)+dA 100 #M BU(10)+ dA 200 #M
80 80
6.7+0.04 7.4+0.05
60 60
83.45-2.14 94.45-2.11
50 50
84.65-2.21 95.85-2.68
8 9
BU(10) + T 100/~ M BU(10) + T 200/xM
80 80
10.1 + 0.07 12.15-0.06
60 60
35.3 + 2.71 39.1+2.14
50 40
37.4 5-1.98 41.1+2.49
BU, BrdU; 10/~g/ml.
82 TABLE 5 EFFECTS OF THYMIDINE (T) AND FLUORODEOXYURIDINE (FU) ON SCE IN NORMAL (KS86), BS2.2 AND BS-SY4 CELLS Expts.
KS86 Cells counted
BS2.2 SCEs + S.E.
BS.SYa
Cells counted
SCEs + S.E.
Cells counted
SCEs 5: S.E.
BU(10) a
80
5.1 + 0.04
60
70.6 + 2.09
60
69.9 + 2.01
BU(10) + FU(0.1/*g/ml) BU(10) + FU(0.4 ~ g/ml)
40 40
9.9+0.05 11.4+0.06
40 40
71.8+2.48 71.7+2.37
40 40
70.65:2.41 71.1_+2.51
BU(10) + T(100/xM) BU(10) + T(200/xM)
80 80
10.1+0.07 12.1+0.06
60 60
35.3+2.71 39.1_+2.14
50 40
37.4+1.98 41.15:2.49
BU(10) + T(100/,M) + FU(0.1/~ g/ml) BU(10) + T(100 ~M) + FU(0.4/~ g/ml) BU(10) + T(200/~M) + FU(0.1 btg/ml) BU(10) + T(200/tM) + FU(0.4/~ g/ml)
40 40 40 40
12.2 + 0.06 13.75:0.07 13.75:0.05 14.15:0.08
40 40 35 35
35.9 5:2.49 36.1 5:2.66 38.45:2.33 39.85:2.45
40. 40 40 40
38.2 5:2.42 38.1 5:2.39 39.45:2.55 42.15:2.39
a BU, BrdU; 10/~g/ml.
pair of complementation was observed in the present experiment. Fig. 3 shows the hybrid metaphase of BrdU-labeled BS2.2 and non-labeled BSSY4, showing the original increased level of SCE in a BS2.2 cell. This strongly suggests that there may be no complementation group in BS.
Effects of nucleosides and FrdU on BS SCE The effect of dC, dG, dA and T on normal and BS SCEs was measured using KS86, BS1.2 (BrdUsensitive) and BS-SY4 (BrdU-resistant) (Table 4). In normal cells, T increased SCEs from 5.1 baseline level up to 12.1 SCEs/cell (average); dA and d G caused a small increase in SCEs, whereas dC showed no effect (Table 4). However, in BS cells (BSa_z, BS-SY4) T produced a significant reduction in SCE from a baseline of 70 to 35 SCEs/cell (Table 4). Table 5 shows the effect of FrdU, in the presence and absence of T, on SCE in normal (KS86) and BS (BS2_2, BS-SY1) cells. FrdU significantly increased SCE in normal cells but produced no noticeable effect on BS SCEs. T plus FrdU produced a slightly higher SCE level in normal cells but made no difference in BS SCE compared to T alone. This strongly suggests that BS has a T pool deficiency. Deoxycytidine caused an SCE reduction from approximately 70 to approximately 60 SCEs/cell. However, both purine deoxyribonucleosides, dA and dG, caused signifi-
cant increase of SCE in BS2. 2 and BS-SY4 cells (Table 4). Discussion Our study of EBV-transformed BS B-LCLs, in which every cell exhibits the characteristically elevated SCE frequency, shows that (1) there are two types (BrdU-sensitive and BrdU-resistant) of cellular proliferation in the BrdU-labeled culture condition; BS-SY4, 5 cells proliferate like normal cells when BrdU is added to their medium, and (2) cell fusion between BrdU-sensitive and BrdU-resistant BS B-LCLs exhibited no complementation, retaining original increased SCE levels, whereas when any of the BS cells labeled with BrdU were fused with non-labeled normal cells, the hybrid cells had almost a normal level of SCE at the first mitosis after fusion. The data presented confirm our earlier observation (Shiraishi et al., 1981) that fusion of BrdU-labeled BS cells with non-labeled normal cells results in correction of the SCE level, indicating that BS cells may have a missing or defective section of D N A which is readily complemented by cell fusion. The origin and the mechanism of high SCE frequency in BS cells grown in BrdU, as compared with normal cells, are obscure. This study is also an attempt to test the contributions of the de-
83
oxynucleosides on the excess SCEs in BS cells, since DNA nucleoside pool bias is a reasonable interpretation of the co-cultivation experiments (Bamezai and Shiraishi, 1986). Addition of individual nucleosides (dA, dC, dG and T) to the growth medium caused significantly similar effects on the SCE frequency in all BS B-LCLs examined. Deoxycytidine addition showed no effect on SCEs in normal cells but decreased SCEs in BS cells from the baseline level of 70 SCEs/cell to about 60 SCEs/cell. Deoxycytidine is known to counteract the increase in SCEs by excess thymidine and BrdU in normal cells (Davidson et al., 1980; Kaufman, 1986). Purine deoxyribonucleosides (dG and dA) caused a significant concentration-dependent increase in SCE frequency both in normal and BS cells (Table 4). Although dT caused a 2-fold increase in normal SCEs, BS SCEs were inhibited about 50%, in both BrdU-sensitive and -resistant BS cells. Addition of FrdU did not greatly affect BS SCEs in the presence of BrdU alone and BrdU plus T. These observations suggest that BS cells with high SCE character may have a low thymidine pool compared with normal cells, which could also account for the spontaneous mutator effect of BS cells and perhaps also a more efficient BrdU substitution in DNA thus potentiating the template effect on SCEs. If this were the case, then the co-cultivation effect (see above) could be due to the transfer of a thymidine metabolite from normal into BS cells through gap-junctions. Direct pool measurements are necessary to test this hypothesis (experiments in progress) which could possibly lead to the elucidation of the molecular defect in BS cells.
Acknowledgements This work was supported in part by a grant-inaid from the Ministry of Education, Science and Culture of Japan, and from the Nissan Science Foundation. The author is grateful to Miss Michiyo Ozawa for her technical assistance.
References Bamezai, R., and Y. Shiraishi (1986) Cell cycle progression and SCE rate of Bloom syndrome cells with/without co-cultivation in the presence/absence of normal cells, Exp. Cell Res., 164, 163-173. Bamezai, R., and Y. Shiraishi (1987) Cell cycle rate and sister
chromatid exchange profile in polyethylene glycol-exposed/unexposed Bloom syndrome and normal cells, Hum. Genet., 75, 356-358. Bartram, C.R., H.W. Rudiger and E. Passarge (1979) Frequency of sister chromatid exchanges in Bloom syndrome fibroblasts reduced by co-cultivation with normal cells, Hum. Genet., 46, 331-334. Chaganti, R.S.K., S. Schonberg and J. German (1974) A manyfold increase in sister chromatid exchanges in Bloom's syndrome lymphocytes, Proc. Natl. Acad. Sci. (U.S.A.), 71, 4508-4512. Davidson, R.L., E.R. Kaufman, C.P. Dougherty, A.M. Ouellette, C.M. Difolco and S.A. Latt (1980) Induction of sister chromatid exchanges by BUdR is largely independent of the BUdR content of DNA, Nature (London), 284, 75-76. German, J. (1969) Bloom's syndrome, I. Genetical and clinical observations in the first twenty-seven patients, Am. J. Hum. Genet., 21, 196-227. Kaufman, E.R. (1986) Induction of sister chromatid exchanges by the replication of 5-bromouracil-substituted DNA under conditions of nucleotide-pool imbalance, Mutation Res., 163, 41-50. Ray, J.H., and J. German (1984) Bloom's syndrome and EM9 cells in BrdU-contalning medium exhibit similarly elevated frequencies of sister chromatid exchange but dissimilar amounts of cellular proliferation and chromosome disruption, Chromosoma, 90, 383-388. Rudiger, H.W., C.R. Bartram, W. Harder and E. Passarge (1980) Rate of sister chromatid exchanges in Bloom syndrome fibroblasts reduced by co-cultivation with normal fibroblasts, Am. J. Hum. Genet., 32, 150-157. Shiraishi, Y., A.F. Freeman and A.A. Sandberg (1976) Increased sister chromatid exchange in bone marrow and blood cells from Bloom's syndrome, Cytogenet. Cell Genet., 17, 162-173. Shiraishi, Y., S. Matsui and A.A. Sandberg (1981) Normalization by cell fusion of sister chromatid exchange in Bloom syndrome lymphocytes, Science, 212, 820-822. Shiraishi, Y., T.H. Yosida and A.A. Sandberg (1982) Analysis of single and twin sister chromatid exchanges in endoreduplicated normal and Bloom syndrome B-lymphoid cells, Chromosoma, 87, 1-8. Shiraishi, Y., I. Kubonishi and A.A. Sandberg (1983a) Establishment of B-lymphoid cell lines retaining cytogenetic characteristics of Bloom syndrome, Cancer Genet. Cytogenet., 56, 129-138. Shiraishi, Y., T.H. Yosida and A.A. Sandberg (1983b) Analyses of bromodeoxyuridine-associated sister chromatid exchanges (SCEs) in Bloom syndrome based on cell fusion: Single and twin SCEs in endoreduplication, Proc. Natl. Acad. Sci. (U.S.A.), 80, 4369-4373. Shiraishi, Y., S. Yosimoto, I. Miyoshi, N. Kondo, T. Orii and A.A. Sandberg (1983c) Dimorphism of sister chromatid exchange in Bloom syndrome B- and T-cell lines trans: formed with Epstein-Barr and adult T-cell leukemia viruses, Cancer Res., 43, 3836-3840. Van Buul, P.P.W., A.T. Natarajan and E.A.M. Verdegaal-Immerzeel (1978) Suppression of the frequencies of sister chromatid exchanges in Bloom's syndrome fibroblasts by co-cultivation with Chinese hamster cells, Hum. Genet., 44, 187-189.