Background: When DNA double-strand breaks (DSB) are induced by ionizing radiation (IR) in cells, histone H2AX is quickly phosphorylated into γ-H2AX (p-S139) around the DSB site. The necessity of DNA-PKcs in regulating the phosphorylation of H2AX in response to DNA damage and cell cycle progression was investigated. Results: The level of γH2AX in HeLa cells increased rapidly with a peak level at 0.25 - 1.0 h after 4 Gy γ irradiation. SiRNA-mediated depression of DNA-PKcs resulted in a strikingly decreased level of γH2AX. An increased γH2AX was also induced in the ATM deficient cell line AT5BIVA at 0.5 - 1.0 h after 4 Gy γ rays, and this IR-increased γH2AX in ATM deficient cells was dramatically abolished by the PIKK inhibitor wortmannin and the DNA-PKcs specific inhibitor NU7026. A high level of constitutive expression of γH2AX was observed in another ATM deficient cell line ATS4. The alteration of γH2AX level associated with cell cycle progression was also observed. HeLa cells with siRNA-depressed DNA-PKcs (HeLa-H1) or normal level DNA-PKcs (HeLa-NC) were synchronized at the G1 phase with the thymidine double-blocking method. At ~5 h after the synchronized cells were released from the G1 block, the S phase cells were dominant (80%) for both HeLa-H1 and HeLa-NC cells. At 8 - 9 h after the synchronized cells released from the G1 block, the proportion of G2/M population reached 56 - 60% for HeLa-NC cells, which was higher than that for HeLa H1 cells (33 - 40%). Consistently, the proportion of S phase for HeLa-NC cells decreased to ~15%; while a higher level (26 - 33%) was still maintained for the DNA-PKcs depleted HeLa-H1 cells during this period. In HeLa-NC cells, the γH2AX level increased gradually as the cells were released from the G1 block and entered the G2/M phase. However, this γH2AX alteration associated with cell cycle progressing was remarkably suppressed in the DNA-PKcs depleted HeLa-H1 cells, while wortmannin and NU7026 could also suppress this cell cycle related phosphorylation of H2AX. Furthermore, inhibition of GSK3β activity with LiCl or specific siRNA could up-regulate the γH2AX level and prolong the time of increased γH2AX to 10 h or more after 4 Gy. GSK3β is a negative regulation target of DNA-PKcs/Akt signaling via phosphorylation on Ser9, which leads to its inactivation. Depression of DNA-PKcs in HeLa cells leads to a decreased phosphorylation of Akt on Ser473 and its target GSK3β on Ser9, which, in other words, results in an increased activation of GSK3β. In addition, inhibition of PDK (another up-stream regulator of Akt/GSK3β) by siRNA can also decrease the induction of γH2AX in response to both DNA damage and cell cycle progression. Conclusion: DNA-PKcs plays a dominant role in regulating the phosphorylation of H2AX in response to both DNA damage and cell cycle progression. It can directly phosphorylate H2AX independent of ATM and indirectly modulate the phosphorylation level of γH2AX via the Akt/GSK3 β signal pathway.

Mitochondrion is an important organelle of eukaryotic cells and a critical target of ionizing radiation (IR) outside the nucleus. The biological significance and mechanism of the non-target effect originating from mitochondria have received much attention in the field of radiation biology and protection. In this study, we investigated the effect, role, and radioprotective significance of cytosolic mitochondrial DNA (mtDNA) and its associated cGAS signaling on hematopoietic injury induced by IR in vitro culture cells and in vivo total body irradiated mice in this study. The results demonstrated that γ-ray exposure increases the release of mtDNA into the cytosol to activate cGAS signaling pathway, and the voltage-dependent anion channel (VDAC) may contribute to IR-induced mtDNA release. VDAC1 inhibitor DIDS and cGAS synthetase inhibitor can alleviate bone marrow injury and ameliorate hematopoietic suppression induced by IR via protecting hematopoietic stem cells and adjusting subtype distribution of bone marrow cells, such as attenuating the increase of the F4/80⁺ macrophage proportion in bone marrow cells. The present study provides a new mechanistic explanation for the radiation non-target effect and an alternative technical strategy for the prevention and treatment of hematopoietic acute radiation syndrome.

The poly(ADP‐ribose) polymerase (PARP) family consists of 17 members of nicotinamide adenine dinucleotide (NAD⁺)‐dependent enzymes that regulate key biological processes by catalyzing adenosine diphosphate (ADP)‐ribosylation, either poly(ADP‐ribosyl)ation (PARylation) or mono(ADP‐ribosyl)ation (MARylation). These biological processes encompass DNA repair, metabolism, telomere maintenance, and immune responses. Based on structural and functional features, the PARP family is classified into subcategories, such as DNA‐dependent PARPs, Tankyrase, CCCH‐type PARPs, MacroPARPs, and atypical PARPs. These enzymes dynamically maintain genome stability through mechanisms, including base excision repair and homologous recombination, while also regulating telomere dynamics and metabolic pathways. Dysregulation of PARP activity is implicated in the pathogenesis of diverse human diseases. Though PARP inhibitors have gained therapeutic interest in oncology, their wider roles in nononcological conditions, such as neurodegenerative diseases, cardiovascular disorders, and viral infections, remain poorly defined. This review elucidates the unique structural features of PARP family members and describes their multiple roles under physiological and pathological conditions, thus providing insights into treatment strategies. Additionally, it summarizes the advances and challenges in PARP‐targeted therapies and explores future directions for innovative therapeutic approaches. The findings may serve as a valuable resource for informing both clinical research and drug development.

Colon cancer stem cells (CSCs), which are highly capable of self‑renewal and proliferation, are involved in colon tumorigenesis and response to therapy. CD133 is considered the most robust surface marker for colorectal cancer stem cells. Although the TP53 gene is frequently mutated in colon cancer, it remains not fully understood whether and how tumor protein p53 (p53) is associated with CD133 expression in colon cancer cells. In the present study, the expression of the CSC biomarker CD133 was investigated in terms of p53 status in colorectal carcinoma HCT116 cells. p53 wild‑type HCT116 (HCT116 p53+/+) and depleted HCT116 (HCT116 p53‑/‑) cells were used throughout this study. Cells carrying the CSC biomarkers CD133 and CD44 were examined by flow cytometry. A dual‑luciferase reporter assay was employed to further confirm the transcriptional regulation of the CD133 promoter by p53. The results demonstrated that there was a significant difference in the % of CD133‑positive cells between the HCT116 p53+/+ cell line (84.84±0.05%) and the HCT116 p53‑/‑ cell line (4.13±0.02%). The mRNA expression levels of CD133 in HCT116 p53+/+ cells were also significantly higher compared with HCT116 p53‑/‑ cells. Knockdown of p53 by specific small interfering RNA greatly reduced the expression of CD133 in HCT116 p53+/+ cells. Transcription factor binding site analysis indicated that there are several p53 binding elements in the CD133 promoter region. A dual‑luciferase reporter assay further demonstrated the transcriptional activation of CD133 promoter by p53. In conclusion, these results suggest that p53 positively regulates the expression of CSC marker CD133 in the HCT116 human colon colorectal cancer cell line. p53 may be involved in the initiation and maintenance of colorectal cancer stem cells through regulating the expression of CD133.

Background: Radiotherapy, a primary approach in cancer treatment, damages normal cells while targeting cancer cells. Therefore, it is crucial to identify drugs with minimal side effects, high reliability, and radioprotective effects to develop novel radiotherapy strategies. Hemerocallis citrina extracts (HCE), which are derived from plants with medicinal and culinary applications, possess antioxidative and anticancer properties. Methods: In this study, we investigated the radioprotective effects of HCE on LO2 cells exposed to radiation to determine whether these effects were mediated through the nuclear factor erythroid 2–related factor 2-cystine–glutamate antiporter/glutathione peroxidase 4 pathway. Results: Cell proliferation experiments demonstrated the radioprotective effect of HCE on LO2 cells. Western blot analysis revealed that HCE regulated B-cell lymphoma protein 2-associated X, Cleaved-caspase 3, and B-cell lymphoma protein 2, thereby inhibiting radiation-induced apoptosis, which was consistent with the flow cytometry results. Conclusions: Moreover, the detection of ferroptosis-related markers indicated that HCE alleviated radiation-induced ferroptosis in LO2 cells through the nuclear factor erythroid 2–related factor 2-cystine–glutamate antiporter/glutathione peroxidase 4 pathway. These findings provide a theoretical basis for the radioprotective effects of HCE on LO2 cells and offer new insights into the development of radioprotective drugs.