The Fate Of Micronuclei And Micronucleated Cells

Micronuclei are closely associated with DNA damage. The presence ofmicronuclei in cells is a common phenomenon following ionizing radiations. Thelevel of micronucleation has been demonstrated to be correlated with theradiosensitivity of tumor cells in many cancers and has been used to predict prognosisafter radiotherapy in many cancers. However, the intrinsic mechanisms for thiscorrelation are poorly characterized due to lack of systematic studies on the fates ofmicronucleated cells because of the limitations of conventional cytogenetictechnologies.In this study, a radioresistant nasopharyngeal carcinoma cell line, CNE1, and aradiosensitive cell line, CNE2, were chosen as a model to investigate the fate ofmicronucleated cells because they can be tracked effectively by long-term live cellimaging due to their good morphology and moderate movement. We generated cellsthat stably express human histone H2B labeled with red fluorescent protein (mCherry)and took extensive serial images of them to record the destination of micronucleatedcells post X-irradiation. Then, the fate of cells was investigated by reverseexamination of these time-lapse records.After retro-analysis of the time-lapse videos, we determine the fates ofirradiation-induced micronucleated cells. Initially, significantly more micronucleiwere observed in radiosensitive cells than in radioresistent cells post irradiation.Additionally, cells with micronuclei were observed to be more likely to die or undergocell cycle arrest when compared with cells without micronuclei post irradiation, andthe more micronuclei the cells contained the more likely they would die or undergocell cycle arrest. Furthermore, micronucleated cells showed predisposition to producedaughter cells with micronuclei through chromosome lagging rather thanchromosomal bridge breaking during cell division. Fluorescence in situ hybridizationusing human pan-centromeric probes revealed that approximately 70% of thesemicronuclei and lagging chromosomes did not contain centromeric signals. Finally,DNA damage was more severe and the p38 stress kinase activity was higher inmicronucleated cells than in micronucleus-free cells as shown by phospho-H2AX andphospho-p38 immunofluorescence staining.Altogether, our observations indicated that the presence of micronuclei coupledwith activated DNA damage response could compromise the proliferation capacity of irradiated cells, providing the evidence and justification for using micronucleus indexas a valuable biomarker of radiosensitivity. Aneuploidy, an abnormal chromosome number, is the major cause ofmiscarriages and mental retardation in humans and is a common characteristic ofmany cancers. Merotelically attached chromosomes (single kinetochore attached bymicrotubules from both spindle pole) lagging in anaphase are considered to be closelyassociated with aneuploidy in daughter cells. Besides, chromosomes lagging duringanaphase and entrapped in micronuclei are considered to be a sign of genetic materialloss and closely associated with aneuploidy formation. However, the contributions ofthe lagging chromosomes to aneuploidy formation are still controversial. Some ofthese assumptions were based on analysis of fixed cells and the actual behaviors oflagging chromosomes and micronuclei in live cells are poorly characterized.To avoid the shortcomings associated with fixed samples, we previously labeledall the chromosomes with fluorescent protein for live cell imaging and have providedpreliminary data on the formations of lagging chromosomes, micronuclei and theireffect on the fate of cells (as we discussed in Part I). Unfortunately, labeling allchromosomes prevents us from precisely tracking the fates of lagging chromosomesand micronuclei. To address these issues new experimental strategies are required.In this study, colorectal cancer HCT116 cells were used to examine the fates ofcells containing lagging chromosomes or micronuclei. We generated derivative cloneswith all the chromosomes labeled with red fluorescent protein (by expression of themCherry-human histone 2B fusing gene) and one chromosome labeled with greenfluorescent protein (GFP), which is achieved by integrating the Escherichia coli Lacoperator (LacO) sequence into a single chromosomal locus in cells expressing theGFP-Lac repressor (LacI) fusing gene. Thus, the localization and dynamics of thesingle GFP-tagged chromosomes in every phase of cell cycle could be monitored inlive cellsHerein, we use live cell imaging to track the fates of merotelically attachedlagging chromosomes labeled with LacI-GFP. We show that most of the laggingchromosomes ultimately migrate to the correct pole of spindle and often formmicronuclei in the daughter cells. However, the chromosomes entrapped inmicronuclei in correct cells do not impair the proliferation of the host cells.Importantly, when the micronucleated cells divide, the chromosomes in micronucleicondense, congregate onto metaphase plate and segregate normally as those in themain nuclei, and consequently result in two daughter cells without micronuclei. Altogether, our results demonstrate that most lagging chromosomes do not result inaneuploidy and corroborate the notion that micronucleus formation does notinevitably lead to loss of genetic materials.