EPAS1 prevents telomeric damage-induced senescence by enhancing transcription of TRF1, TRF2, and RAD50.

Telomeres are nucleoprotein structures located at the end of each chromosome, which function in terminal protection and genomic stability. Telomeric damage is closely related to replicative senescence in vitro and physical aging in vivo. As relatively long-lived mammals based on body size, bats display unique telomeric patterns, including the up-regulation of genes involved in alternative lengthening of telomeres (ALT), DNA repair, and DNA replication. At present, however, the relevant molecular mechanisms remain unclear. In this study, we performed cross-species comparison and identified EPAS1, a well-defined oxygen response gene, as a key telomeric protector in bat fibroblasts. Bat fibroblasts showed high expression of EPAS1, which enhanced the transcription of shelterin components TRF1 and TRF2, as well as DNA repair factor RAD50, conferring bat fibroblasts with resistance to senescence during long-term consecutive expansion. Based on a human single-cell transcriptome atlas, we found that EPAS1 was predominantly expressed in the human pulmonary endothelial cell subpopulation. Using in vitro-cultured human pulmonary endothelial cells, we confirmed the functional and mechanistic conservation of EPAS1 in telomeric protection between bats and humans. In addition, the EPAS1 agonist M1001 was shown to be a protective compound against bleomycin-induced pulmonary telomeric damage and senescence. In conclusion, we identified a potential mechanism for regulating telomere stability in human pulmonary diseases associated with aging, drawing insights from the longevity of bats.

RETSAT associates with DDX39B to promote fork restarting and resistance to gemcitabine based chemotherapy in pancreatic ductal adenocarcinoma.

BACKGROUND: Severe hypoxia is a prominent character of pancreatic ductal adenocarcinoma (PDAC) microenvironment. In the process of gemcitabine based chemotherapy, PDAC cells are insulted from replication stresses co-induced by hypoxia and gemcitabine. However, PDAC cells get outstanding abilities to resist to such harsh conditions and keep proliferating, causing a major obstacle for current therapy. RETSAT (Retinol Saturase) is defined as a hypoxia convergent gene recently, with high expression in PDAC hypoxic sectors. This study aimed to explore the roles of RETSAT in replication stress resistance and hypoxia adaptation in PDAC cells, and decipher the underlying mechanism. METHODS: The expression of RETSAT was examined in TCGA (The Cancer Genome Atlas), human pancreatic cancer microarray, clinical specimens and cell lines. Functions of RETSAT were studied by means of DNA fiber assay and comet assay in monolayer cultured PDAC cell lines, three dimensional spheroids, patient derived organoids and cell derived xenograft mouse models. Mechanism was investigated by using iPOND (isolate proteins on nascent DNA) combined with mass spectrometry, immunoprecipitation and immunoblotting. RESULTS: First, we found the converse relationship of RETSAT expression and PDAC chemotherapy. That is, PDAC patients with high RETSAT expression correlated with poor survival, while ones holding low RETSAT expression were benefitted more in Gemcitabine based chemotherapy. Second, we identified RETSAT as a novel replication fork associated protein. HIF-1α signaling promotes RETSAT expression under hypoxia. Functionally, RETSAT promoted fork restarting under replication stress and maintained genomic stability. Third, we uncovered the interaction of RETSAT and R-loop unwinding helicase DDX39B. RETSAT detained DDX39B on forks to resolve R-loops, through which avoided fork damage and CHK1 initiated apoptosis. Targeting DDX39B using chemical CCT018159 sensitized PDAC cells and organoids to gemcitabine induced apoptosis, highlighting the synergetic application of CCT018159 and gemcitabine in PDAC chemotherapy. CONCLUSIONS: This study identified RETSAT as a novel replication fork protein, which functions through interacting with DDX39B mediated R-loop clearance to promote fork restarting, leading to cellular resistance to replication stresses co-induced by tumor environmental hypoxia and gemcitabine in pancreatic ductal adenocarcinoma.

[Effect of germacrone in alleviating HUVECs damaged by H2O2-induced oxidative stress].

This study focuses on the protective effect of germacrone on human umbilical vein endothelial cells(HUVECs) damaged by H2O2-induced oxidative stress and its possible mechanisms. The oxidative damage model was established by using 500 µmol•L⁻¹ H2O2 to treat HUVECs for 3 hours, and then protected with different concentrations of germacrone for 24 hours. The effect of germacrone on cell viability of HUVECs damaged by H2O2 was detected by MTT. The contents of PGI2, TXB2, ET-1, t-PA, PAI-1, TNF-α and IL-6 were detected by ELISA. The content of NO was detected by using nitrate reductase method. Colorimetry was used to detect NOS and GSH-Px. The contents of MDA, SOD and LDH were detected by TBA, WST-1 and microplate respectively. Apoptosis was observed by Hoechst 33258 fluorescent staining. The mRNA expressions of Bax, Bcl-2 and Caspase-3 in cells were detected by RT-PCR. The results showed that the cell damage rate was 52% after treated with 500 µmol•L⁻¹ H2O2 for 3 hours. The cell activity was increasing with the rise of germacrone concentration within the range of 20-200 mol•L⁻¹. Compared with normal group, the contents of PGI2, NO, T-NOS, t-PA, SOD, GSH-Px and Bcl-2 mRNA expressions were lower after damaged with H2O2. The contents of PAI-1, ET-1, IL-6, TNF-α, TXB2, LDH, MDA, Bax mRNA and Caspase-3 mRNA expressions were increased. Compared with model group, the contents of PGI2, NO, T-NOS, t-PA, SOD, GSH-Px and Bcl-2 mRNA expressions were increased after treated with germacrone. The contents of PAI-1, ET-1, IL-6, TNF-α, TXB2, LDH, MDA, Bax mRNA and Caspase-3 mRNA expressions were lower after treated with germacrone. According to Hoechst 33258 fluorescence staining, compared with normal group, the cell membrane and the nucleus showed strong dense blue fluorescence, and the number of cells significantly decreased in model group. Compared with model group, blue fluorescence intensity decreased in drug group. The above findings demonstrate that germacrone may improve the effect on HUVECs damaged by H2O2-induced oxidative stress by resisting oxidation and inhibiting cell apoptosis.