The hematopoietic system is highly radiosensitive; thus therapeutic and unwanted irradiation can produce significant/lethal acute and delayed bone marrow injury. In a murine hematopoietic-acute radiation syndrome (H-ARS) model, a single dose of 16,16 dimethyl Prostaglandin E2 (dmPGE2) given 15 min to 3 hrs before lethal (LD50/30-90/30) total body irradiation (TBI) results in 100% survival of mice at 30 days, with accelerated recovery of WBC, and hematopoietic stem and progenitor cell number and function.
To explore mechanisms responsible for the radio-protective effect(s) of dmPGE2 we evaluated hematopoietic cells at early points post-TBI, focusing on oxidative pathways and epigenetic changes. Hematopoietic cells were isolated and evaluated at 1 hr and 1, 3 and 9 days post LD50 [853 cGy] TBI. Mitochondrial ROS and membrane potential were measured by flow cytometry in SLAM-SKL cells. Protein levels of gH2AX, LC3b, BNIP3, P16, P53, P62 acetylated H3K9 and H4K16 histones and SIRT1 were measured by flow cytometry. Acetylation of H3K9 and H4K16 at the KU70, Ku80, LC3b, Atg5, Atg7, Atg12, BNIP3, NF-kB and p53 promoters was evaluated by ChIP.
Lethal radiation significantly increased HSC mitochondrial ROS and reduced membrane potential at 1,3 and 9 days post-TBI. Significant DNA damage was observed at 1 hour as measured by gH2AX. In contrast, mitochondrial ROS and DNA damage (gH2AX) were significantly lower in mice that received dmPGE2 before irradiation. In vehicle treated irradiated mice, we observed hyper-acetylated H3K9 and H4K16 at the Ku70/80 promoter, suggesting diminished capacity of Ku70/80 to repair DNA damage. In contrast, in mice treated with dmPGE2, H3K9 and H4K16 acetylation was significantly lower. Reduced mitochondrial membrane potential seen in vehicle treated mice was also prevented. The senescence markers SA-beta-gal and P16 in HSC were significantly reduced in PGE2 treated mice.
The sirtuin family NAD-dependent deacetylase, SIRT1, has broad functions including gene silencing and DNA repair. In control irradiated mice SIRT1 levels dramatically decreased over 9 days post-irradiation, but remained significantly higher in dmPGE treated mice, which may account for the low acetylation levels of H3K9 and H4K16 in PGE treated mice. Consistent with elevated SIRT1 in PGE2 treated mice, we also observed global deacetylation of H3K9 and H4K16 in lineage negative and SLAM LSK cells at 1-3 days post-TBI. P53 is a key transcriptional regulator of genes involved in cell cycle, apoptosis and DNA repair and is acetylated after DNA damage. In control mice, irradiation resulted in robust acetylation of histone proteins at the p53 promoter, whereas dmPGE2 administration prevented acetylation of H4K16, but not H3K9. In addition, dmPGE2 blocked p53-mediated apoptosis via repression of p53 transcription.
Since SIRT1 promotes autophagy, we evaluated autophagy related genes. CHIP analyses revealed low levels of acetylated-H3K9 at the LC3B, ATG7 and NF-KB promoters and deacetylated H4K16 at the LC3B, ATG7, BNIP3 and NF-kB promoters in PGE treated mice compared to vehicle controls. No effects on histone deacetylation of the Atg5 and Atg12 genes. BNIP3 is a mitophagy marker that when elevated downregulates mitochondrial mass and respiration. In irradiated control mice, BNIP3 levels were decreased at 1through 9 days, but remained elevated in mice treated with dmPGE2. The autophagy substrate P62 was significantly increased at 1 and 3 days post-irradiation in control mice, but was significantly lower in PGE2 treated mice. SIRT1 activation also leads to decreased binding of NFkB-p65 to DNA and attenuated oxidative stress through reduced transcription. CHIP data showed that TBI increased acetylation of H3K9 and H4K16 at the NF-kB p65 promoter in vehicle treated mice, whereas hyperacetylation of H3K9 and H4K16 and NF-kB transcription did not occur in mice treated with dmPGE2 before irradiation.
These studies suggest that the radio protective effect of dmPGE2 and accelerated recovery of hematopoiesis following radiation exposure is mediated in part through reduction in oxidative stress and DNA damage induced in HSC and epigenetically, through prevention of histone acetylation of genes associated with DNA repair, autophagy and mitophagy, likely mediated through elevation of SIRT1.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.