Levels Of DNA Damage In Cells Research Articles

Promotion of Cervical Cancer Cell Proliferation by miR-130b Expression Level Changes and Inhibition of its Apoptosis by Targeting CDKN1A Gene.

Background: Dysregulation of miR-130b expression is associated with the development of different cancers. However, the description of the biological roles of miR-130b in the growth and survival of cervical cancer cells is limited. Methods: The miR-130b levels in cervical cancer cells during different stages of growth were determined using reverse transcription-quantitative PCR. The methylation level of DNA sequences upstream of the miR-130b gene was measured using an SYBR Green-based quantitative methylation-specific PCR. Reverse transcription-quantitative PCR, Western blotting, and fluorescence report assays were used to identify the miR-130b-targeted gene. Cell counting kit-8 and comet assays were used to determine cell viability and DNA damage levels in cells, respectively. EdU Apopllo488 in vitro Flow Cytometry kit, propidium iodide staining, anti-γ-H2AX antibody staining, and Annexin-V apoptosis kit were subsequently used to determine DNA synthesis rates, cell cycle distribution, count of DNA double-strand breaks, and levels of apoptotic cells. Results: miR-130b levels increased at exponential phases of the growth of cervical cancer cells but reduced at stationary phases. The methylation of a prominent CpG island near the transcript start site suppressed the miR-130b gene expression. MiR-130b increased cell viability, promoted both DNA synthesis and G1 to S phase transition of the cells at exponential phases, but reduced cell viability accompanied by accumulations of DNA breaks and augmentations in apoptosis rates of the cells in stationary phases by targeting cyclin-dependent kinase inhibitor 1A mRNA. Conclusion: miR-130b promoted the growth of cervical cancer cells during the exponential phase, whereas it impaired the survival of cells during stationary phases.

High Expression of BCAT1 promotes Acute Myeloid Leukemia Progression and Sensitize to PARP Inhibitor

Acute myeloid leukemia (AML) is a highly heterogeneous disease with poor clinical prognosis, especially to cytogenetically normal AML(CN-AML)patients, which belong to moderate prognosis group. The clinical outcomes of them are not consistent. Therefore, further exploration of novel biomarkers and development of new anti-tumor drugs is urgently needed. Recently, accumulating evidence have emerged and demonstrated that branched-chain amino acids (BCAA) are essential for tumor growth and proliferation. Branched-chain aminotransferase 1(BCAT1), a BCAA metabolic enzyme, which correlates with cancer aggression, not only that, it has been reported that overexpression of BCAT1in leukemia cells decreased intracellular αKG levels, showed the characteristics of stem cells and displayed a phenotype like cases carrying IDH mutations. By limiting intracellular αKG, BCAT1high expression AML cells prone to cause an increase in DNA damage probably via suppressing homologous recombination. We firstly analyzed the prognostic significance of BCAT1expression in CN-AML patients. Collectively, we found that patients with high levels of BCAT1had poor prognosis, the median overall survival time is shorter in BCAT1high group (356 days versus 570 days)(Figure A). Next, we performed gene knockout in THP-1 and MV4-11 cells with relatively high expression of BCAT1and gene overexpressing in the relatively low expression cell line OCI-AML3. In summary, BCAT1overexpression contributed to cell growth, whereas suppression of BCAT1markedly limited cell proliferation and colony-forming ability by blocking cell circle at G0/G1 phase(Figure B and C).The mice model we conducted further validated the role of BCAT1expression in vitro. Notably, we found that knockout and overexpression of BCAT1can respectively reduce and enhance the tumor burden(Figure D)and effects the time of survival(Figure E). Because both BCAT1high expression and IDH mutation could decrease αKG levels and suppress αKG dependent dioxygenases, it is tempting to speculate that there is a possibility of a "BRCAness" phenotype when BCAT1is overexpressed.We indeed observed a rise in the base line level of DNA damage marker γ-H2AX in BCAT1overexpressing cell line(Figure F). In addition, the sensitivity of cells to cisplatin is also positively correlated with BCAT1expression levels, the IC50 of BCAT1overexpressing cell line OCI-AML3 is much lower than control. Consistently, knocking out of BCAT1decreased sensitivity to cisplatin. So we further tested the relationship between PARPi talazoparib(BMN 673) and BCAT1expression, Use a gain of function approach we increased susceptibility to PARPi, together with the loss of function data, these results strongly suggest that BCAT1promotes cell sensitivity to PARPi(Figure G). We believe that the combination of high expression of BCAT1and PARPi produces synthetic lethality. In addition, the ability to trap PARP1 of PARPi may produce unacceptable toxicity when combined with conventional doses of cytotoxic chemotherapies. So, we next combined PARP inhibitors with DNA-damaging agent daunorubicin, the synergistic effect is promising on BCAT1overexpressed OCI-AML3 cell especially at high dose, however, the synergistic effect of the control group is weak, sometimes even manifested as antagonistic.Consistent with previous reports, IDH1/2mutAML is vulnerable to PARP inhibition as monotherapy, but especially when combined with daunorubicin treatment. These results also confirmed our hypothesis that BCAT1overexpression mimics the phenotype of IDHmutcells, not only as DNA hypermethylation, but also leads to increased DNA damage levels in cells, increasing sensitivity to PARP inhibitor and DNA-damaging drugs. Through qPCR and WB assays, we found the same trend: DNA damage response-related protein ATM was down-regulated after BCAT1overexpression, which preliminarily explained why DNA damage was increased after BCAT1overexpression. After exogenous supplementation with the aKG analog DM-aKG, the enhancement of DNA damage caused by overexpression of BCAT1can be reversed. In summary, this study indicated that CN-AML patients with high BCAT1expression had poor prognosis. BCAT1plays the role as oncogene, can induce DNA damage that renders AML cells sensitive to PARP inhibition and DNA damage agents, and can be used as a novel therapeutic option for AML. Figure Disclosures No relevant conflicts of interest to declare.

Applications of the comet assay in particle toxicology: air pollution and engineered nanomaterials exposure.

Exposure to ambient air particles is associated with elevated levels of DNA strand breaks (SBs) and endonuclease III, formamidopyrimidine DNA glycosylase (FPG) and oxoguanine DNA glycosylase-sensitive sites in cell cultures, animals and humans. In both animals and cell cultures, increases in SB and in oxidatively damaged DNA are seen after exposure to a range of engineered nanomaterials (ENMs), including carbon black, carbon nanotubes, fullerene C60, ZnO, silver and gold. Exposure to TiO2 has generated mixed data with regard to SB and oxidatively damaged DNA in cell cultures. Nanosilica does not seem to be associated with generation of FPG-sensitive sites in cell cultures, while large differences in SB generation between studies have been noted. Single-dose airway exposure to nanosized carbon black and multi-walled carbon nanotubes in animal models seems to be associated with elevated DNA damage levels in lung tissue in comparison to similar exposure to TiO2 and fullerene C60. Oral exposure has been associated with augmented DNA damage levels in cells of internal organs, although the doses have been typically very high. Intraveneous and intraperitoneal injection of ENMs have shown contradictory results dependent on the type of ENM and dose in each set of experiments. In conclusion, the exposure to both combustion-derived particles and ENMs is associated with increased levels of DNA damage in the comet assay. Particle size, composition and crystal structure of ENM are considered important determinants of toxicity, whereas their combined contributions to genotoxicity in the comet assay are yet to be thoroughly investigated.