Response of PRIMPOL-Knockout Human Lung Adenocarcinoma A549 Cells to Genotoxic Stress.

Human DNA primase/polymerase PrimPol synthesizes DNA primers de novo after replication fork stalling at the sites of DNA damage, thus contributing to the DNA damage tolerance. The role of PrimPol in response to the different types of DNA damage is poorly understood. We knocked out the PRIMPOL gene in the lung carcinoma A549 cell line and characterized the response of the obtained cells to the DNA damage caused by hydrogen peroxide, methyl methanesulfonate (MMS), cisplatin, bleomycin, and ionizing radiation. The PRIMPOL knockout reduced the number of proliferating cells and cells in the G2 phase after treatment with MMS and caused a more pronounced delay of the S phase in the cisplatin-treated cells. Ionizing radiation at a dose of 10 Gy significantly increased the content of apoptotic cells among the PRIMPOL-deficient cells, while the proportion of cells undergoing necroptosis increased in both parental and knockout cells at any radiation dose. The viability of PRIMPOL-deficient cells upon the hydrogen peroxide-induced oxidative stress increased compared to the control cells, as determined by the methyl tetrazolium (MTT) assay. The obtained data indicate the involvement of PRIMPOL in the modulation of adaptive cell response to various types of genotoxic stress.

The formation of DNA single-strand breaks and alkali-labile sites in human blood lymphocytes exposed to 365-nm UVA radiation.

The potency of UVA radiation, representing 90% of solar UV light reaching the earth's surface, to induce human skin cancer is the subject of continuing controversy. This study was undertaken to investigate the role of reactive oxygen species in DNA damage produced by the exposure of human cells to UVA radiation. This knowledge is important for better understanding of UV-induced carcinogenesis. We measured DNA single-strand breaks and alkali-labile sites in human lymphocytes exposed ex vivo to various doses of 365-nm UV photons compared to X-rays and hydrogen peroxide using the comet assay. We demonstrated that the UVA-induced DNA damage increased in a linear dose-dependent manner. The rate of DNA single-strand breaks and alkali-labile sites after exposure to 1J/cm(2) was similar to the rate induced by exposure to 1 Gy of X-rays or 25 µM hydrogen peroxide. The presence of either the hydroxyl radical scavenger dimethyl sulfoxide or the singlet oxygen quencher sodium azide resulted in a significant reduction in the UVA-induced DNA damage, suggesting a role for these reactive oxygen species in mediating UVA-induced DNA single-strand breaks and alkali-labile sites. We also showed that chromatin relaxation due to hypertonic conditions resulted in increased damage in both untreated and UVA-treated cells. The effect was the most significant in the presence of 0.5M Na(+), implying a role for histone H1. Our data suggest that the majority of DNA single-strand breaks and alkali-labile sites after exposure of human lymphocytes to UVA are produced by reactive oxygen species (the hydroxyl radical and singlet oxygen) and that the state of chromatin may substantially contribute to the outcome of such exposures.