The fate of micronucleated cells post X-irradiation detected by live cell imaging

Abstract

Micronuclei are closely related to DNA damage. The presence of micronuclei in mammalian cells is a common phenomenon post ionizing radiation. The level of micronucleation in tumor cells has been used to predict prognosis after radiotherapy in many cancers. In order to understand how irradiation-induced micronuclei affect cell fate, we performed extensive long-term live cell imaging on X-irradiated nasopharyngeal carcinoma (NPC) cells. To visualize the dynamics of micronuclei more clearly, chromosomes were stably labeled with red fluorescent protein (RFP) by targeting to human histone H2B. Initially, significantly more micronuclei were observed in radiosensitive cells than in radioresistant cells post irradiation. Additionally, cells with micronuclei were found to be more likely to die or undergo cell cycle arrest when compared with micronucleus-free cells after irradiation, and the more micronuclei the cells contained the more likely they would die or undergo arrest. Moreover, micronucleated cells showed predisposition to produce daughter cells with micronuclei through chromosome lagging. Fluorescence in situ hybridization using human pan-centromeric probes revealed that about 70% of these micronuclei and lagging chromosomes did not contain centromeric signals. Finally, DNA damage was more severe and p38 stress kinase activity was higher in micronucleated cells than in micronucleus-free cells as shown by phospho-H2AX and phospho-p38 immunofluorescence staining. Altogether, our observations indicated that the presence of micronuclei coupled with activated DNA damage response could compromise the proliferation capacity of irradiated cells, providing the evidence and justification for using micronucleus index as a valuable biomarker of radiosensitivity.

Introduction

Micronuclei are small, DNA/chromatin-containing structures in the cytoplasm of cells. They show similar texture, staining and morphology to that of the main nuclei of the same cells [1], [2]. The occurrence of micronuclei has been used as an indicator of either the clastogenic or aneugenic effects of chemical or physical agents [3], [4]. They have also been used to predict increased risk of pregnancy complications [5], cancer risk [6], [7] and cardiovascular mortality [8] if the micronucleus frequency is high. Results from several studies suggest that the micronucleus assay can be used to determine radiosensitivity of cells and predict radiotherapy outcomes [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]. However, although micronucleation was found to be correlated with radiosensitivity, the underlying mechanisms of this correlation are still poorly defined. This is mainly due to the lack of systematic studies on the dynamic fates of micronucleated cells after irradiation because of limitations of traditional methods based on the analysis of fixed cells.

Time-lapse imaging has proven to be an effective tool for investigating cell migration, cell division, differentiation, and organelle or chromosome dynamics [20], [21]. Live observations on individual cells provide a unique opportunity to track transient dynamic cellular processes that cannot be detected in fixed specimens [20]. By expressing fluorescent protein coupled proteins, cell structures can be tracked at high resolution in real time [22]. As micronuclei are composed of chromosome fragments or whole chromosomes, labeling histone H2B with fluorescent protein makes it possible to scrutinize dynamics of micronuclei in live cells. Using this labeling technique, the mechanisms of micronucleus formation have been explored previously [2], [23], [24], [25], [26], [27], and very interesting results have been published. However, the fate of micronuclei and the fate of cells harboring micronuclei are largely unknown. In order to address these issues, we here performed long-term live cell imaging to track the dynamics of micronuclei and micronucleated cells post X-irradiation.

Nasopharyngeal carcinoma (NPC) is a common disease in Southern China and South-East Asia [28]. Like many cancers, radiation therapy is still the main treatment modality for NPC [29]. In this preliminary study, a radioresistant NPC cell line, CNE1, and a radiosensitive cell line, CNE2 [30], [31], were chosen as a model to investigate the fate of micronucleated cells because they can be tracked effectively by long-term live cell imaging due to their good morphology and moderate movement. We generated cells that stably express histone 2B (H2B) labeled with red fluorescent protein (RFP) and took extensive serial images of them to record the destination of micronucleated cells post X-irradiation. Then, the fate of cells was investigated by reverse examination of these time-lapse records.