Scoring of SCE frequency per cell cycle in CHO chromosomes by means of a standardized 3-way-differential staining method

Abstract

We have developed 2 alternative protocols to obtain 3-way differentiation (TWD) in Chinese hamster ovary (CHO) cells, in order to analyze the sister-chromatid exchange (SCE) frequency on a per-generation basis. In protocol A, 5-bromodeoxyuridine (BrdU) substitution into DNA was low during the first S period (S1) and high during the next 2 (S2 and S3). In protocol B, on the other hand, BrdU substitution was high during S1 and low during S2 and S3. The main advantage of our procedures is the high efficiency and reproducibility reached by controlling the relative incorporation of BrdU and deoxythymidine (dT) into replicating DNA in the presence of 5-fluorodeoxyuridine (FdU) throughout 3 consecutive S periods. The comparison of the results obtained for both protocols allowed us to evaluate the role of BrdU in the induction of SCEs in each cell cycle. Our results from M3 chromosomes seem to indicate that, under our experimental conditions, neither the concentration of BrdU in the medium nor the rate of BrdU incorporation into nascent DNA is the basic feature responsible for SCE formation. Indeed, the experiments reported here seem to demonstrate that the nature of parental DNA is crucial for the formation of the BrdU-induced SCEs.