Chemotherapy (Etoposide) Induced Intermingling of Hetero-Chromatin and Euchromatin Compartments in Senescent PA-1 Embryonal Carcinoma Cells

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Often, neoadjuvant therapy, which relies on the induction of double-strand breaks (DSBs), is used to shrink tumor by cell apoptosis, before surgery to trigger cancer cell apoptosis. However, recent studies have suggested that this treatment may also induce a fluctuating state between senescence and stemness in PA-1 embryonal carcinoma cells, potentially affecting therapeutic outcomes. Thus, the respective epigenetic pathways are up or downregulated over a time period of days. These fluctuations go hand in hand with changes in the spatial DNA organization. By means of Single Molecule Localization Microscopy in combination with mathematical evaluation tools for pointillist data sets, we investigated the organization of euchromatin and heterochromatin on the nano-scale on the third and fifth day after the etoposide treatment. Using fluorescently labeled antibodies against H3K9me3 (heterochromatin tri-methylation sites) and H3K4me3 (euchromatin tri-methylation sites), we found that the induction of DSBs led to de-condensation of heterochromatin and compaction of euchromatin, with a peak effect on day 3 after the treatment. On day 3 we also observed co-localization of euchromatin and heterochromatin marks which usually occur in exclusive low-overlapping network-like compartments. The evaluation of the SMLM data by topological tools (persistent homology and persistent imaging) and principal component analysis, as well as confocal microscopy analysis of H3K9me3 and H3K4me3 stained PA-1 cells supported the findings of distinct shifts in euchromatin and heterochromatin organization in a subpopulation of these cells during the days after the treatment. Furthermore, by means of flow cytometry, it was shown that the rearrangements in the chromatin organization coincided with simultaneous upregulation of OCT4A and SOX2, stemness promoters, and p21Cip1 and p27, senescence promoters. Our findings suggest potential applications in improving cancer therapy by inhibiting chromatin remodeling and thus preventing therapy-induced senescence.

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