A novel role for the mono-ADP-ribosyltransferase PARP14/ARTD8 in promoting homologous recombination and protecting against replication stress.

Genomic instability, a major hallmark of cancer cells, is caused by incorrect or ineffective DNA repair. Many DNA repair mechanisms cooperate in cells to fight DNA damage, and are generally regulated by post-translational modification of key factors. Poly-ADP-ribosylation, catalyzed by PARP1, is a post-translational modification playing a prominent role in DNA repair, but much less is known about mono-ADP-ribosylation. Here we report that mono-ADP-ribosylation plays an important role in homologous recombination DNA repair, a mechanism essential for replication fork stability and double strand break repair. We show that the mono-ADP-ribosyltransferase PARP14 interacts with the DNA replication machinery component PCNA and promotes replication of DNA lesions and common fragile sites. PARP14 depletion results in reduced homologous recombination, persistent RAD51 foci, hypersensitivity to DNA damaging agents and accumulation of DNA strand breaks. Our work uncovered PARP14 as a novel factor required for mitigating replication stress and promoting genomic stability.

Transcriptome Analysis of FEN1 Knockdown HEK293T Cell Strain Reveals Alteration in Nucleic Acid Metabolism, Virus Infection, Cell Morphogenesis and Cancer Development.

AIM AND OBJECTIVE: Flap endonuclease-1 (FEN1) plays a central role in DNA replication and DNA damage repair process. In mammals, FEN1 functional sites variation is related to cancer and chronic inflammation, and supports the role of FEN1 as a tumor suppressor. However, FEN1 is overexpressed in multiple types of cancer cells and is associated with drug resistance, supporting its role as an oncogene. Hence, it is vital to explore the multi-functions of FEN1 in normal cell metabolic process. This study was undertaken to examine how the gene expression profile changes when FEN1 is downregulated in 293T cells. MATERIALS AND METHODS: Using the RNA sequencing and real-time PCR approaches, the transcript expression profile of FEN1 knockdown HEK293T cells have been detected for the next step evaluation, analyzation, and validation. RESULTS: Our results confirmed that FEN1 is important for cell viability. We showed that when FEN1 downregulation led to the interruption of nucleic acids related metabolisms, cell cycle related metabolisms are significantly interrupted. FEN1 may also participate in non-coding RNA processing, ribosome RNA processing, transfer RNA processing, ribosome biogenesis, virus infection and cell morphogenesis. CONCLUSION: These findings provide insight into how FEN1 nuclease might regulate a wide variety of biological processes, and laid the foundation for understanding the role of other RAD2 family nucleases in cell growth and metabolism.

FEN1 inhibitor increases sensitivity of radiotherapy in cervical cancer cells.

BACKGROUND: Cervical cancer is one of the most common causes of cancer-associated mortality among affected women in the world. At present, treatment with weekly cisplatin plus ionizing radiation (IR) therapy is the standard regimen for cervical cancer, especially for locally advanced cervical cancer. The purpose of this study is to determine whether FEN1 inhibitors could enhance the therapeutic effect of IR therapy. METHODS: Western blot was applied to determine the expression of FEN1- and apoptosis-related proteins. Cell growth inhibition assay and colony formation assay were used to determine the effects of FEN1 inhibitor and IR exposure for Hela cells in vitro. CRISPR technology was used to knockdown FEN1 expression level of 293T cells, and tumor xenograft in nude mice was employed to determine the effects of FEN1 inhibitor and IR exposure on tumor growth in vivo. RESULTS: Our data revealed that FEN1 is overexpressed in HeLa cell and can be upregulated further by IR. We also demonstrated that FEN1 inhibitor enhances IR sensitivity of cervical cancer in vitro and in vivo. CONCLUSION: FEN1 inhibitor SC13 could sensitize radiotherapy of cervical cancer cell.

Effects and mechanism of amyloid ß1-42 on mitochondria in astrocytes.

Amyloid ß (Aß)1-42 is strongly associated with Alzheimer's disease (AD). The effects of Aß1­42 on astrocytes remain largely unknown. The present study focused on the effects of Aß1­42 on U87 human glioblastoma cells as astrocytes for in vitro investigation and mouse brains for in vivo investigation. The mechanism and regulation of mitochondria and cytochrome P450 reductase (CPR) were also investigated. As determined by MTT assays, low doses of Aß1­42 (<1 µM) marginally promoted astrocytosis compared with the 0 µM group within 24 h, however, after 48 h treatment these doses reduced cellular growth compared with the 0 µM group. Furthermore, Aß1­42 doses >5 µM inhibited the growth of U87 cells compared with the 0 µM group after 24 and 48 h treatment. Immunofluorescence analysis demonstrated that astrocytosis was also observed in early stage AD mice compared with wild­type (WT) mice. In addition, concentrations of Aß1­42 were also significantly higher in early stage AD mice compared with WT mice, however, the levels were markedly lower compared with later stage AD mice, as determined by ELISA. In addition to increased levels of Aß1­42 in mice with later stage AD, reduced astrocyte staining was observed compared with WT mice. Western blotting indicated that the effect of Aß1­42 on U87 cell apoptosis may be regulated via Bcl­2 and caspase­3 located in mitochondria, whose functions, including adenosine triphosphate generation, electron transport chain and mitochondrial membrane potential, were inhibited by Aß1­42. During this process, the expression and activity of cytochrome P450 reductase was also downregulated. The current study provides novel insight into the effects of Aß1­42 on astrocytes and highlights a potential role for astrocytes in the protection against AD.