Transcriptome Analysis of Cells Exposed to Actinomycin D and Nutlin-3a Reveals New Candidate p53-Target Genes and Indicates That CHIR-98014 Is an Important Inhibitor of p53 Activity

• Published in: International journal of molecular sciences Vol. 22; no. 20; p. 11072
• Main Authors: Łasut-Szyszka, Barbara, Małachowska, Beata, Gdowicz-Kłosok, Agnieszka, Krześniak, Małgorzata, Głowala-Kosińska, Magdalena, Zajkowicz, Artur, Rusin, Marek
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 14-10-2021; MDPI
• Subjects: Actinomycin; Adaptor Proteins, Signal Transducing - genetics; Adaptor Proteins, Signal Transducing - metabolism; Alzheimer's disease; Aminopyridines - chemistry; Aminopyridines - metabolism; Aminopyridines - pharmacology; Apolipoprotein E; Apolipoproteins E - genetics; Apolipoproteins E - metabolism; Apoptosis; Apoptosis - drug effects; BLNK protein; Cancer therapies; Cell activation; Cell cycle; Cell Line, Tumor; CRISPR; Cytokines; Dactinomycin - pharmacology; Enzyme inhibitors; Exposure; Gene expression; Gene Expression Regulation - drug effects; Genes; GSK-3; Humans; Hypotheses; Imidazoles - pharmacology; Immune system; Innate immunity; Interferon; Interferon regulatory factor 1; Interferon Regulatory Factor-1 - genetics; Interferon Regulatory Factor-1 - metabolism; kinase inhibitor; Kinases; Lung cancer; Mutagenesis, Site-Directed; p53; p53 Protein; Piperazines - pharmacology; Polymerase chain reaction; Promoter Regions, Genetic; Proteins; Pyrimidines - chemistry; Pyrimidines - metabolism; Pyrimidines - pharmacology; Radiation; RNA Interference; RNA, Small Interfering - metabolism; RNA-Seq; Signal transduction; Transcriptomes; Tumor Suppressor Protein p53 - antagonists & inhibitors; Tumor Suppressor Protein p53 - genetics; Tumor Suppressor Protein p53 - metabolism; Western blotting; kinase inhibitor; GSK-3; innate immunity; RNA-Seq; p53
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms222011072

Co-treatment with actinomycin D and nutlin-3a (A + N) strongly activates p53. Previously we reported that CHIR-98014 (GSK-3 kinase inhibitor), acting in cells exposed to A + N, prevents activation of
-an innate immunity and p53-regulated gene associated with Alzheimer's disease. In order to find novel candidate p53-target genes and genes regulated by CHIR-98014, we performed RNA-Seq of control A549 cells and the cells exposed to A + N, A + N with CHIR-98014 or to CHIR-98014. We validated the data for selected genes using RT-PCR and/or Western blotting. Using CRISPR/Cas9 technology we generated p53-deficient cells. These tools enabled us to identify dozens of candidate p53-regulated genes. We confirmed that p53 participates in upregulation of
,
and
.
assists in activation of immune cells,
codes for apolipoprotein associated with Alzheimer's disease and
is activated by interferon gamma and regulates expression of antiviral genes. CHIR-98014 prevented or inhibited the upregulation of a fraction of genes stimulated by A + N. Downregulation of GSK-3 did not mimic the activity of CHIR-98014. Our data generate the hypothesis, that an unidentified kinase inhibited by CHIR-98014, participates in modification of p53 and enables it to activate a subset of its target genes, e.g., the ones associated with innate immunity.