Fate of micronuclei and micronucleated cells after treatment of HeLa cells with different genotoxic agents

Abstract

Although micronuclei are well-known biomarkers of genotoxic damage, the biological consequences of micronucleus induction are only poorly understood. To further elucidate these consequences, HeLa cells stably expressing histone 2B coupled with green fluorescent protein were used for long-term live cell imaging to investigate the fate of micronuclei and micronucleated cells after treatment of cells with various genotoxic agents (doxorubicin (20, 30 and nM), tert-butyl hydroperoxide (tBHP, 50, 100 and 150 µM), radiation (0.5, 1 and 2 Gy), methyl methanesulfonate (MMS, 20, 25 and 30 µg/ml) and vinblastine (1, 2 and 3 nM)). Most micronuclei persist for multiple cell cycles or reincorporate while micronucleated cells were more prone to cell death, senescence and fatal mitotic errors compared to non-micronucleated cells, which is consistent with previous studies using etoposide. No clear substance-related effects on the fate of micronuclei and micronucleated cells were observed. To further investigate the fate of micronuclei, extrusion of micronuclei was studied with treatments reported as inducing the extrusion of micronuclei. Since extrusion was not observed in HeLa cells, the relevance of extrusion of micronuclei remains unclear. In addition, degradation of micronuclei was analysed via immunostaining of γH2AX, which demonstrated a high level of DNA damage in micronuclei compared to the main nuclei. Furthermore, transduction with two reporter genes (LC3B-dsRed and LaminB1-dsRed) was conducted followed by long-term live cell imaging. While autophagy marker LC3B was not associated with micronuclei, Lamin B1 was found in approximately 50% of all micronuclei. While degradation of micronuclei was not observed to be a frequent fate of micronuclei, the results show impaired stability of DNA and micronuclear envelope indicating rupture of micronuclei as a pre-step to chromothripsis.

Mechanism and role of nuclear laminin B1 in cell senescence and malignant tumors

“…Although micronucleus is a well-known biomarker of genotoxic damage, little is known regarding the biological consequences of micronucleus induction. Reimann et al [ 39 ] found that the micronuclei did not share a common fate after treating HeLa cells with different genetic agents; however, DNA stability and micronucleus envelope were impaired, and LMNB1 was expressed in approximately 50% of the micronuclei. This finding indicated that micronucleus breakdown is required for chromosome fragmentation, while the damaged DNA is retained in the cell, which may promote tumor development [ 39 ].…”

“…Reimann et al [ 39 ] found that the micronuclei did not share a common fate after treating HeLa cells with different genetic agents; however, DNA stability and micronucleus envelope were impaired, and LMNB1 was expressed in approximately 50% of the micronuclei. This finding indicated that micronucleus breakdown is required for chromosome fragmentation, while the damaged DNA is retained in the cell, which may promote tumor development [ 39 ]. Nuclear membrane rupture is known to be associated with the loss or defect of lamin; however, the key physical determinants of nuclear membrane rupture remain unknown.…”