THE CENTROMERE AS A TARGET FOR THE INDUCTION OF CHROMOSOME-DAMAGE IN RESTING AND PROLIFERATING MAMMALIAN-CELLS - ASSESSMENT OF MITOMYCIN C-INDUCED GENETIC-DAMAGE AT KINETOCHORES AND CENTROMERES BY A MICRONUCLEUS TEST IN MOUSE SPLENOCYTES

The cytokinesis-block micronucleus assay (MN) on murine splenocytes wa s used for the estimation of chromosome damage in a resting cell popul ation in vivo that can be induced to proliferate in vitro, Mitomycin C at different doses (10(-8), 6 x 10(-8), 10(-7), 6 x 10(-7) and 10(-6) M) was used to induce cytogenetic damage in resting and cycling splen ocytes, Antikinetochore antibodies (CREST) and two-colour fluorescence irt situ hybridization (FISH) with minor and major satellite DNA were applied. These approaches allowed the detailed characterization of th e mechanisms by which MN originates, since it was possible to identify breaks induced in pericentric heterochromatin (resulting in MN contai ning the major but not the minor satellite DNA) or detachment/disrupti on of kinetochore (resulting in different frequencies of MN containing kinetochore or both probes). Based on the evidence that resting and c ycling mouse splenocytes are characterized by different spatial distri bution of centromeric regions, the hypothesis was tested that the dama ge induced by mutagens at centromeres is influenced by the phase of th e cell cycle in which the cells are treated. Data presented here show that resting and cycling splenocytes are both sensitive to mitomycin C action, and indicate that this compound has an aneugenic potential, b esides its strong clastogenic activity. In particular, results obtaine d after CREST and FISH characterization of MN differed when cells were treated during proliferation, suggesting a disruption/detachment of k inetochores induced by mitomycin C at this cell stage. Furthermore, un der the same treatment condition the proportion of MN containing the m ajor satellite DNA only was greater than expected on the basis of rand om breakage at this site, Treatment of resting cells produced aneugeni c damage, but without evidence of disruption/detachment of kinetochore s or preferential breakage at the centromere. These results indicate t hat the amount and type of chromosome damage induced by mitomycin C in mouse splenocytes differ in relation to the proliferative status of t reated cells.