Shiraishi Y, Li M J
Department of Anatomy, Kochi Medical School, Japan.
Mutat Res. 1990 Jun;230(2):177-86. doi: 10.1016/0027-5107(90)90055-9.
The effect of bromodeoxyuridine (BrdU)-substituted DNA template and thymidine (dT) pool on excess sister-chromatid exchanges (SCEs) was studied in Bloom syndrome (BS) cells and an ataxia telangiectasia (AT)-derived mutant cell line (AsHa). When BS endomitotic cells were labeled with low and high (or high and low) BrdU concentrations during S1 and S2, only the BrdU concentration during S1 phase affected the observed SCE. In BS cells about a 10-fold increase in SCEs occurs during or following replication on a BrdU-substituted template (high-high and high-low BrdU labeling) relative to the normal DNA template. SCEs decreased to about half in AsHa cells labeled with various BrdU doses (40, 60, 80 and 100 micrograms/ml) during only S1, compared with those labeled during S1 and S2. Co-cultivation of AsHa and BS cells resulted in a significant reduction in SCE level from 70 to 13-17 in BS cells, lowered the BrdU concentrations necessary for sister-chromatid differential (SCD) staining from 40 to 10 micrograms/ml with normal SCE level and resulted in decreased level of SCEs at high BrdU concentrations (80-100 micrograms/ml) (12-14 SCE) in AsHa cells, compared with the originally increased SCE level (36.65 SCE at 100 micrograms/ml) without co-culture. However, co-cultivation between AsHa and normal cells lowered the BrdU dose necessary for SCD staining from 40 to 30 micrograms/ml; the dT pool possibly balanced at this level, which is clearly higher than that at co-cultivation between AsHa and BS cells. The reason for the very high BrdU doses needed to achieve SCD would seem to be that AsHa cells have high levels of thymidylate (TMP) synthetase, which maintain a large endogenous thymidine pool. This has been confirmed by direct measurement. These findings strongly support that excess and decreased dT pools are closely related to the condition necessary for high SCE induction.
研究了溴脱氧尿苷（BrdU）取代的DNA模板和胸苷（dT）库对布卢姆综合征（BS）细胞和共济失调毛细血管扩张症（AT）衍生的突变细胞系（AsHa）中姐妹染色单体交换（SCE）过量的影响。当BS内有丝分裂细胞在S1和S2期分别用低浓度和高浓度（或高浓度和低浓度）的BrdU标记时，只有S1期的BrdU浓度会影响观察到的SCE。在BS细胞中，相对于正常DNA模板，在BrdU取代的模板上复制期间或之后（高高和高低BrdU标记），SCE增加约10倍。与仅在S1期标记的细胞相比，在S1和S2期均用不同剂量BrdU（40、60、80和100微克/毫升）标记的AsHa细胞中，SCE减少至约一半。AsHa细胞与BS细胞共培养导致BS细胞的SCE水平从70显著降低至13 - 17，使姐妹染色单体差异（SCD）染色所需的BrdU浓度从40微克/毫升降至10微克/毫升，且SCE水平正常，并且与未共培养时最初增加的SCE水平（100微克/毫升时为36.65 SCE）相比，AsHa细胞在高BrdU浓度（80 - 100微克/毫升）下的SCE水平降低（12 - 14 SCE）。然而，AsHa细胞与正常细胞共培养使SCD染色所需的BrdU剂量从40微克/毫升降至30微克/毫升；此时dT库可能处于平衡状态，这明显高于AsHa细胞与BS细胞共培养时的水平。实现SCD所需的BrdU剂量非常高的原因似乎是AsHa细胞具有高水平的胸苷酸（TMP）合成酶，可维持大量内源性胸苷库。这已通过直接测量得到证实。这些发现有力地支持了过量和减少的dT库与高SCE诱导所需条件密切相关。
