Pro-inflammatory RNA:DNA Hybrids Are p53 Independently Boosted by Hyperbaric Oxygen: a Subcellular Distribution Analysis by Automated Quantitative Imaging

Abstract

Purpose RNA:DNA hybrids are co-transcriptional products with acknowledged cytoplasmic pro-inflammatory role as activators of the cGAS-STING pathway. We recently proved them also as radiation-induced senescence messages for the abscopal effect mediation, demonstrating the need for a functional p53 for their production and release in A549 and H1299 tumour cells. However, little is known about their role under different stress conditions, especially in cancer cells. Methods In this work, we open the investigation making use of automated quantitative imaging to characterize the hybrid subcellular distribution in HeLa cells grown under different oxygen pressures or exposed to different ionizing radiation doses. After cell imaging by confocal fluorescent microscopy, we apply automated imaging methods developed on purpose to quantify hybrid foci and nuclear cluster intensity, regional and local density and dimension. Results We show that alteration of culture oxygenation increases hybrid cytoplasmic presence, especially when caused by an hyperoxic environment, with evident hybrid gathering at the cell membrane. Ionizing radiations always fail to increase hybrids, in accordance with the absence of functional p53 in HeLa cells. However, dose-dependent effects are still evident and suggest a threshold dose of 7.5 Gy for remarkable hybrid reduction. Conclusion Together with our previous results, these data demonstrate for the first time that different types of stress can increase hybrid production in cancer cells and by at least two different pathways, one p53-dependent triggerable by ionizing radiations and one p53-independent triggerable by oxidative stress. Together, our findings provide a starting point for understanding hybrid role in tumour stress response.

Maternal exposure to hyperbaric oxygen at the preimplantation stages increases apoptosis and ectopic Cdx2 expression and decreases Oct4 expression in mouse blastocysts via Nrf2‐Notch1 upregulation and Nf2 downregulation

“…Previous studies investigating the effects of HBO‐ or H 2 O 2 ‐induced oxidative stress on human osteoblastic, HeLa, and RPE‐1 cells have reported the occurrence of single‐stranded DNA damage and RNA:DNA hybrids 61–63 . These molecular events were associated with increased activation of ATM, formation of Tp53bp1 foci, and induction of apoptosis 61–63 .…”

“…Previous studies investigating the effects of HBO-or H 2 O 2 -induced oxidative stress on human osteoblastic, HeLa, and RPE-1 cells have reported the occurrence of single-stranded DNA damage and RNA:DNA hybrids. [61][62][63] These molecular events were associated with increased activation of ATM, formation of Tp53bp1 foci, and induction of apoptosis. [61][62][63] Consistent with these studies, we observed increased Tp53bp1 expression at both the protein and mRNA levels in the HBO-exposed blastocysts (Figures 4K,L and 5E).…”

“…[61][62][63] These molecular events were associated with increased activation of ATM, formation of Tp53bp1 foci, and induction of apoptosis. [61][62][63] Consistent with these studies, we observed increased Tp53bp1 expression at both the protein and mRNA levels in the HBO-exposed blastocysts (Figures 4K,L and 5E). We detected Tp53bp1-positive and active Caspase-3-positive nuclei in the ICM but not in the TE of the HBO-exposed blastocysts (Figure 3A,C-E).…”

“…59,60 These foci are implicated in the detection of single-and double-stranded DNA fragmentation, as well as pro-inflammatory RNA:DNA hybrids that are induced by HBO exposures. [61][62][63] However, there was a scarcity of studies examining the effects of HBO on DNA damage and cell apoptosis in preimplantation embryos.…”

“…Additionally, Nrf2 has been reported to stimulate the transcription of the gene encoding tumor protein p53 binding protein 1 (Tp53bp1), which forms intranuclear foci of DNA damage in association with the histone protein γH2AX 59,60 . These foci are implicated in the detection of single‐ and double‐stranded DNA fragmentation, as well as pro‐inflammatory RNA:DNA hybrids that are induced by HBO exposures 61–63 . However, there was a scarcity of studies examining the effects of HBO on DNA damage and cell apoptosis in preimplantation embryos.…”