Pessina A, Bonomi A, Casati S, Collotta A, Croera C, Marafante E, Palitti F, Gribaldo L
Department of Public Health, Microbiology, Virology, University of Milan, Milan, Italy.
Cell Prolif. 2007 Aug;40(4):568-79. doi: 10.1111/j.1365-2184.2007.00456.x.
The susceptibility of two cell lines, WEHI-3B myelomonocytic leukaemia and its variant Ciprofloxacin-resistant WEHI-3B/CPX to undergo apoptosis induced by Ciprofloxacin was studied and compared.
Apoptosis was checked by measuring the DNA fragmentation and determining the ratio of apoptotic/necrotic cells. The relationship between the induction of apoptosis and G(1), S or G(2) block in the cell cycle has also been investigated and cytogenetical evaluation of chromosomal aberrations in both cell lines has been carried out. The regulation of expression of Bax and Bcl-2 was also checked by western blotting after Ciprofloxacin treatment.
We observed that the resistance of the subline was caused by a small percentage of cells that underwent apoptosis during continuous exposure to Ciprofloxacin in comparison with the parental cell line, whereas the percentage of necrotic cells remained unchanged. The WEHI-3B cells showed a G(2) block and a higher degree of cytogenetic damage after drug exposure. The two cell lines expressed the same level of Bax and Bcl-2 following stimulation by Ciprofloxacin. Only in the resistant subclone, the ratio Bcl-2/Bax reversed in the anti-apoptotic gene expression.
The resistance to ciprofloxacin observed is not related to mitochondrial function and although Bcl-2/Bax ratio behaviour does not fully explain the resistance of the WEHI3B/CPX subclone it is consistent with phenotypic character of resistance to CPX. The toxic effect on sensitive cells could be mediated by the cell cycle arrest whereas in the resistant clone, the prolonged G(2) phase could play a key role to favour cell cycle progression and proliferation.
研究并比较两种细胞系，即WEHI-3B骨髓单核细胞白血病细胞系及其耐环丙沙星变异体WEHI-3B/CPX对环丙沙星诱导凋亡的敏感性。
通过检测DNA片段化及确定凋亡/坏死细胞比例来检查凋亡情况。还研究了凋亡诱导与细胞周期中G(1)、S或G(2)期阻滞之间的关系，并对两种细胞系的染色体畸变进行了细胞遗传学评估。环丙沙星处理后，通过蛋白质免疫印迹法检测Bax和Bcl-2表达的调节情况。
我们观察到，与亲代细胞系相比，亚系的耐药性是由一小部分在持续暴露于环丙沙星期间发生凋亡的细胞引起的，而坏死细胞的比例保持不变。药物暴露后，WEHI-3B细胞表现出G(2)期阻滞和更高程度的细胞遗传学损伤。环丙沙星刺激后，两种细胞系表达相同水平的Bax和Bcl-2。仅在耐药亚克隆中，抗凋亡基因表达中的Bcl-2/Bax比值发生了逆转。
观察到的对环丙沙星的耐药性与线粒体功能无关，尽管Bcl-2/Bax比值行为不能完全解释WEHI3B/CPX亚克隆的耐药性，但它与对CPX的耐药表型特征一致。对敏感细胞的毒性作用可能由细胞周期停滞介导，而在耐药克隆中，延长的G(2)期可能对促进细胞周期进程和增殖起关键作用。
