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Chemotherapy (Etoposide)-Induced Intermingling of Heterochromatin and Euchromatin Compartments in Senescent PA-1 Embryonal Carcinoma Cells
by
, , , , and
Marc Bayer
1,†
,
Jaroslava Zajakina
1,†,
Myriam Schäfer
1,
Kristine Salmina
2,
Felikss Rumnieks
2,
Juris Jansons
2
,
Felix Bestvater
3,
Reet Kurg
4,
Jekaterina Erenpreisa
2
and
Michael Hausmann
1,*
1
Kirchhoff-Institute for Physics, Heidelberg University, Im Neuenheimer Feld 227, 69120 Heidelberg, Germany
2
Latvian Biomedical Research and Study Centre, Rātsupītes iela 1, Rīga LV-1067, Latvia
3
German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
4
Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Cancers 2025, 17(15), 2480; https://doi.org/10.3390/cancers17152480 (registering DOI)
Submission received: 8 June 2025
/
Revised: 13 July 2025
/
Accepted: 23 July 2025
/
Published: 26 July 2025
(This article belongs to the Special Issue Insights from the Editorial Board Member)
Simple Summary
Current cancer chemotherapy uses etoposide to induce DNA double-strand breaks for cell killing, which can cause fluctuations in stemness promoters like OCT4A, SOX-2, or NANOG in relation to senescence promoters p21Cip1, p27, or p16ink4A, and mitigate anti-cancer therapy. These fluctuations take place over several days and are associated with changes in the organization of euchromatin and heterochromatin. Here, we show how the induction of double-strand breaks by etoposide led to the compaction of euchromatin and de-condensation of heterochromatin on day 3. This was accompanied by a co-localization of euchromatin and heterochromatin marks as detected by Single-Molecule Localization Microscopy.
Abstract
Background: Often, neoadjuvant therapy, which relies on the induction of double-strand breaks (DSBs), is used prior to surgery to shrink tumors by inducing 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 spatial DNA organization. Methods: 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 at the nanoscale on the third and fifth day after etoposide treatment. Results: 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 the de-condensation of heterochromatin and compaction of euchromatin, with a peak effect on day 3 after the treatment. On day 3, we also observed the co-localization of euchromatin and heterochromatin, which have marks that usually occur in exclusive low-overlapping network-like compartments. The evaluation of the SMLM data using topological tools (persistent homology and persistent imaging) and principal component analysis, as well as the confocal microscopy analysis of H3K9me3- and H3K4me3-stained PA-1 cells, supported the findings that distinct shifts in euchromatin and heterochromatin organization took place 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 chromatin organization coincided with the simultaneous upregulation of the stemness promotors OCT4A and SOX2 and senescence promotors p21Cip1 and p27. Conclusion: Our findings suggest potential applications to improve cancer therapy by inhibiting chromatin remodeling and preventing therapy-induced senescence.
