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.

Establishment of a mechanism-based in vitro coculture assay for evaluating the efficacy of immune checkpoint inhibitors.

Cancer immunotherapy, which blocks immune checkpoint molecules, is an effective therapeutic strategy for human cancer patients through restoration of tumor-infiltrating (TI) cell function. However, evaluating the efficacy of immune checkpoint inhibitors (ICIs) is difficult because no standard in vitro assay for ICI efficacy evaluation exists. Additionally, blocking a particular immune checkpoint receptor (ICR) is insufficient to restore T cell functionality, because other ICRs still transduce inhibitory signals. Therefore, limiting inhibitory signals transduced via other ICRs is needed to more accurately assess the efficacy of ICIs targeting a particular immune checkpoint. Here, we introduce a newly developed in vitro coculture assay using human peripheral blood mononuclear cells (hPBMCs) and engineered human cancer cell lines. We enriched CD8+ T cells from hPBMCs of healthy donors through low-dose T cell receptor stimulation and cytokine (human IL-2 and IL-7) addition. These enriched CD8+ T cells were functional and expressed multiple ICRs, especially TIM-3 and TIGIT. We also established immune checkpoint ligand (ICL) knockout (KO) cancer cell lines with the CRISPR-Cas9 system. Then, we optimized the in vitro coculture assay conditions to evaluate ICI efficacy. For example, we selected the most effective anti-TIM-3 antibody through coculture of TIM-3+CD8+ T cells with PD-L1-/-PVR-/- cancer cells. In summary, we developed a mechanism-based in vitro coculture assay with hPBMCs and ICL KO cancer cell lines, which could be a useful tool to identify promising ICIs by providing reliable ICI efficacy information.

Definitive localization of intracellular proteins: Novel approach using CRISPR-Cas9 genome editing, with glucose 6-phosphate dehydrogenase as a model.

Studies to determine subcellular localization and translocation of proteins are important because subcellular localization of proteins affects every aspect of cellular function. Such studies frequently utilize mutagenesis to alter amino acid sequences hypothesized to constitute subcellular localization signals. These studies often utilize fluorescent protein tags to facilitate live cell imaging. These methods are excellent for studies of monomeric proteins, but for multimeric proteins, they are unable to rule out artifacts from native protein subunits already present in the cells. That is, native monomers might direct the localization of fluorescent proteins with their localization signals obliterated. We have developed a method for ruling out such artifacts, and we use glucose 6-phosphate dehydrogenase (G6PD) as a model to demonstrate the method's utility. Because G6PD is capable of homodimerization, we employed a novel approach to remove interference from native G6PD. We produced a G6PD knockout somatic (hepatic) cell line using CRISPR-Cas9 mediated genome engineering. Transfection of G6PD knockout cells with G6PD fluorescent mutant proteins demonstrated that the major subcellular localization sequences of G6PD are within the N-terminal portion of the protein. This approach sets a new gold standard for similar studies of subcellular localization signals in all homodimerization-capable proteins.

Changes of Protein Expression after CRISPR/Cas9 Knockout of miRNA-142 in Cell Lines Derived from Diffuse Large B-Cell Lymphoma.

BACKGROUND: As microRNA-142 (miR-142) is the only human microRNA gene where mutations have consistently been found in about 20% of all cases of diffuse large B-cell lymphoma (DLBCL), we wanted to determine the impact of miR-142 inactivation on protein expression of DLBCL cell lines. METHODS: miR-142 was deleted by CRISPR/Cas9 knockout in cell lines from DLBCL. RESULTS: By proteome analyses, miR-142 knockout resulted in a consistent up-regulation of 52 but also down-regulation of 41 proteins in GC-DLBCL lines BJAB and SUDHL4. Various mitochondrial ribosomal proteins were up-regulated in line with their pro-tumorigenic properties, while proteins necessary for MHC-I presentation were down-regulated in accordance with the finding that miR-142 knockout mice have a defective immune response. CFL2, CLIC4, STAU1, and TWF1 are known targets of miR-142, and we could additionally confirm AKT1S1, CCNB1, LIMA1, and TFRC as new targets of miR-142-3p or -5p. CONCLUSIONS: Seed-sequence mutants of miR-142 confirmed potential targets and novel targets of miRNAs can be identified in miRNA knockout cell lines. Due to the complex contribution of miRNAs within cellular regulatory networks, in particular when miRNAs highly present in RISC complexes are replaced by other miRNAs, primary effects on gene expression may be covered by secondary layers of regulation.

The stability and oncogenic function of LIN28A are regulated by USP28.

RNA-binding protein LIN28A is often highly expressed in human malignant tumors and is involved in tumor metastasis and poor prognosis. Knowledge about post-translational regulatory mechanisms governing LIN28A protein stability and function is scarce. Here, we investigated the role of ubiquitination and deubiquitination on LIN28A protein stability and report that LIN28A protein undergoes ubiquitination. Ubiquitin-specific protease 28 (USP28), a deubiquitinating enzyme, interacts with and stabilizes LIN28A protein to extend its half-life. USP28, through its deubiquitinating activity, antagonizes LIN28A protein turnover by reversing its proteasomal degradation. Our study describes the consequential impacts of USP28-mediated stabilization of LIN28A protein on enhancing cancer cell viability, migration and ultimately augmenting LIN28A-mediated tumor progression. Overall, our data suggest that a synergistic, combinatorial approach of targeting LIN28A with USP28 would contribute to effective cancer therapeutics.

Base Excision DNA Repair Deficient Cells: From Disease Models to Genotoxicity Sensors.

Base excision DNA repair (BER) is a vitally important pathway that protects the cell genome from many kinds of DNA damage, including oxidation, deamination, and hydrolysis. It involves several tightly coordinated steps, starting from damaged base excision and followed by nicking one DNA strand, incorporating an undamaged nucleotide, and DNA ligation. Deficiencies in BER are often embryonic lethal or cause morbid diseases such as cancer, neurodegeneration, or severe immune pathologies. Starting from the early 1980s, when the first mammalian cell lines lacking BER were produced by spontaneous mutagenesis, such lines have become a treasure trove of valuable information about the mechanisms of BER, often revealing unexpected connections with other cellular processes, such as antibody maturation or epigenetic demethylation. In addition, these cell lines have found an increasing use in genotoxicity testing, where they provide increased sensitivity and representativity to cell-based assay panels. In this review, we outline current knowledge about BER-deficient cell lines and their use.

Designer Nuclease-Mediated Generation of Knockout THP1 Cells.

Recent developments in the field of designer nucleases allow the efficient and specific manipulation of genomic architectures in eukaryotic cell lines. To this end, it has become possible to introduce DNA double strand breaks (DSBs) at user-defined genomic loci. If located in critical coding regions of genes, thus induced DSBs can lead to insertions or deletions (indels) that result in frameshift mutations and thereby the knockout of the target gene. In this chapter, we describe a step-by-step workflow for establishing knockout cell clones of the difficult-to-transfect suspension cell line THP1. The here described protocol encompasses electroporation, cell cloning, and a deep sequencing-based genotyping step that allows the in-parallel analysis of 96 cell clones per gene of interest. Furthermore, we describe the use of the analysis tool OutKnocker that allows rapid identification of cell clones with all-allelic frameshift mutations.