Analyzing Mitotic Chromosome Structural Defects After Topoisomerase II Inhibition or Mutation
For analyzing chromosome structural defects that result from topoisomerase II (topo II) dysfunction we have adapted classical cell cycle experiments, classical cytological techniques and the use of a potent topo II inhibitor (ICRF-193). In this chapter, we describe in detail the protocols used and we discuss the rational for our choice and for the adaptations applied. We clarify in which cell cycle stages each of the different chromosomal aberrations induced by inhibiting topo II takes place: lack of chromosome segregation, undercondensation, lack of sister chromatid resolution, and lack of chromosome individualization. We also put these observations into the context of the two topo II-dependent cell cycle checkpoints. In addition, we have devised a system to analyze phenotypes that result when topo II is mutated in human cells. This serves as an alternative strategy to the use of topo II inhibitors to perturb topo II function.
Key wordsTopoisomerase II Chromatid resolution Condensation Chromosome individualization ICRF-193 Topo II checkpoint
The chromosome preparation technique was originally devised in Prof. Robert T Johnson’s laboratory (Mammalian Cell DNA Repair Group, University of Cambridge, UK) and has been modified over the years by Drs. Giménez-Abián and Clarke. Silver impregnation was developed for mammalian mitotic chromosomes by Dr. Giménez-Abián, but is based on methods used to stain meiotic grasshopper chromosomes by Rufas et al. [10, 11]. HeLa EM2-11ht cells were provided by Weiderfeld et al. . Development of the protocols described in this chapter was partly dependent on funding from grants BFU2008-03579/BMC and 2008201165 from the VICT (MCI, Spain) and NIH grant CA099033 to DJC.
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