Differences in the origins of kinetochore positive and kinetochore negative micronuclei: A live cell imaging study.
Micronuclei (MNi) are extensively used to evaluate genotoxicity and chromosomal instability. Classification of kinetochore negative (K MNi) and kinetochore positive micronuclei (K+MNi) improves the specificity and sensitivity of the micronucleus (MN) test; however, the fundamental differences in the origins of K MNi and K+MNi have not been addressed due to the limitations of traditional methods. In the current study, HeLa CENP B GFP H2B mCherry cells were constructed in which histone 2B (H2B) and centromere protein B (CENP B) were expressed as fusion proteins to monomeric Cherry (mCherry) and EGFP, respectively. MNi were identified using H2B mCherry; K+MN contained CENP B GFP, while K MN did not. Long term live cell imaging was conducted to examine MN formation in the dual color fluorescent HeLa cells. The results suggested that K MNi were derived from kinetochore negative displaced chromosomes (K DCs) kinetochore negative lagging chromosomes (K LCs) and fragments of broken chromosome bridges (CBs) during late mitotic stages. The results also indicated that K+MNi are derived from kinetochore positive displaced chromosomes (K+DCs) kinetochore positive lagging chromosomes (K+LCs) and fragments of broken CBs. Different aberrant chromosomes emerged during mitosis at different frequencies and developed into K MNi and/or K+MNi in the daughter cells at different rates. K+LCs formed K+MNi at a higher frequency than K+DCs, and K LCs formed K MNi at a higher rate than K DCs; however, broken CBs transformed into K MNi and/or K+MNi. In summary, these results show that K MNi and K+MNi have different origins in HeLa cells and that each mechanism of MN formation contributes differently to the overall number of K MNi and K+MNi.
Mutation research