DISTINCT TEMPORAL PATTERNS OF NUCLEAR AND NUCLEOLAR REMODELING PRECEDING CELL DEATH INDUCED BY CHEMICAL AND PHYSICAL STRESSORS

Abstract

Two major classes of prominently apoptogenic stressors—physical and chemical—were employed to resolve the detailed chronology of chromatin and nucleolar pre-mortal remodeling in HeLa cells stably expressing histone H2B–GFP. Physical damage was induced by ionizing γ-irradiation and non-ionizing heat shock, whereas chemical stress was triggered by the rDNA-intercalating anticancer agents actinomycin D and cisplatin. Actinomycin D served as a classical model of rRNA synthesis inhibition, reliably inducing canonical nucleolar segregation, and was used as a reference standard to compare the chronology and dynamics of nuclear and nucleolar deformations elicited by other stressors. Cisplatin was applied to evaluate whether chemically induced DNA damage produces nucleolar responses comparable to those triggered by physical factors.           
       Long-term live-cell 2D time-lapse imaging was performed to monitor GFP-tagged nucleosomal chromatin behavior, with particular emphasis on intra- and perinucleolar chromatin dynamics. To assess nucleolar remodeling, stress-induced three-dimensional redistribution of the RNA polymerase I–associated transcription factor UBTF was analyzed by immunolabeling followed by 3D reconstruction. Ourresults demonstrate stressor-specific patterns of intranucleolar 3D reorganization of UBTF and nucleolus-associated condensed chromatin. These findings provide structural insights relevant to chemo- and radiotherapy outcomes and may inform strategies aimed at enhancing nucleolar stress, mitotic catastrophe, and apoptosis in resistant malignancies.