Quantitative single-cell analysis of PML-RARα oncogene-induced DNA damage along cell cycle progression

Oncogene activation represents a critical initiating event in tumorigenesis, promoting replication stress and impairing genome stability. The PML-RARα oncogene, responsible for acute promyelocytic leukemia, disrupts the organization of promyelocytic leukemia nuclear bodies (PML-NBs) and alters DNA damage repair mechanisms, thereby contributing to the accumulation of unrepaired DNA lesions. In this study, we employ confocal microscopy to investigate how PML-RARα activation triggers DNA damage in relation to cell cycle progression and DNA replication sites using the inducible U937-PR9 cell model. Specifically, we describe an imaging protocol that provides a quantitative, single-cell analysis of DNA damage along the cell cycle. The protocol is based on an EdU-based sorting strategy in combination with γH2A.X immunolabeling to monitor the accumulation of double-strand breaks (DSBs) across different phases of the cell cycle. Image cross-correlation spectroscopy (ICCS) is used to assess the spatial association between DNA replication foci and DNA damage sites. This approach reveals that PML-RARα expression leads to a general increase in DNA damage compared to the untreated condition, with elevated levels particularly evident in replicating cells. Moreover, the spatial analysis suggests that PML-RARα shifts the colocalization between DNA damage and replication activity toward earlier stages of DNA synthesis. Together, these results demonstrate that our quantitative imaging-based method is well suited to dissect oncogene-induced genome instability throughout the different stages of the cell cycle and in relation to replication foci, highlighting its value as a tool to investigate how replication stress contributes to the build-up of DNA lesions at single-cell resolution.