DNA damage causes TP53-dependent coupling of self-renewal and senescence pathways in embryonal carcinoma cells

Abstract

Recent studies have highlighted an apparently paradoxical link between self-renewal and senescence triggered by DNA damage in certain cell types. In addition, the finding that TP53 can suppress senescence has caused a re-evaluation of its functional role in regulating these outcomes. To investigate these phenomena and their relationship to pluripotency and senescence, we examined the response of the TP53-competent embryonal carcinoma (EC) cell line PA-1 to etoposide-induced DNA damage. Nuclear POU5F1/OCT4A and P21CIP1 were upregulated in the same cells following etoposide-induced G₂M arrest. However, while accumulating in the karyosol, the amount of OCT4A was reduced in the chromatin fraction. Phosphorylated CHK2 and RAD51/γH2AX-positive nuclear foci, overexpression of AURORA B kinase and moderate macroautophagy were evident. Upon release from G₂M arrest, cells with repaired DNA entered mitoses, while the cells with persisting DNA damage remained at this checkpoint or underwent mitotic slippage and gradually senesced. Reduction of TP53 using sh- or si-RNA prevented the upregulation of OCT4A and P21CIP1 and increased DNA damage. Subsequently, mitoses, micronucleation and senescence were all enhanced after TP53 reduction with senescence confirmed by upregulation of CDKN2A/P16INK4A and increased sa-β-galactosidase positivity. Those mitoses enhanced by TP53 silencing were shown to be multicentrosomal and multi-polar, containing fragmented and highly deranged chromosomes, indicating a loss of genome integrity. Together, these data suggest that TP53-dependent coupling of self-renewal and senescence pathways through the DNA damage checkpoint provides a mechanism for how embryonal stem cell-like EC cells safeguard DNA integrity, genome stability and ultimately the fidelity of self-renewal.

Keywords: :

• TP53
• OCT4A/POU5F1
• self-renewal
• tumor cells
• DNA damage
• pluripotency
• senescence

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors would like to thank Professor Hao and Dr. Zhao, Dept. of Histology and Embryology, Shandong University School of Medicine for the kind gift of the OCT4A and OCT4A-Psg-1 vector constructs and Dr. Pawel Zayakin (Latvian Biomedical Centre) for insightful discussions. T.R.J. was funded through an MRC PhD studentship and the Gerald Kerkut Charitable Trust awarded to P.A.T. and M.S.C.; J.E. was funded by the Latvian National Research Programme 2010–2013 “BIOMEDICINE” and AH by European Social Fund within the project “Support for Doctoral Studies at University of Latvia.” Exchange visits between Riga and Southampton were supported by the Royal Society of London. The publishing costs associated with this article are in part provided by the ERDF project no. 2DP/2.1.1.2.0/10/ APIA/VIAA/004.