Cytological Analysis of Chromosome Structural Defects that Result from Topoisomerase II Dysfunction
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 rationale 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 take 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.
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, U.K.) 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. (8, 9). 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 D.J.C.
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