Abstract
RNA synthesis inhibitors and protein synthesis inhibitors are useful for investigating whether biological events with unknown mechanisms require transcription or translation; however, the dependence of RNA synthesis has been difficult to verify because many RNA synthesis inhibitors cause adverse events that trigger a p53 response. In this study, we screened a library containing 9600 core compounds and obtained STK160830 that shows anti-apoptotic effects in irradiated wild-type-p53-bearing human T-cell leukemia MOLT-4 cells and murine thymocytes. In many of the p53-impaired cells and p53-knockdown cells tested, STK160830 did not show a remarkable anti-apoptotic effect, suggesting that the anti-apoptotic activity is p53-dependent. In the expression analysis of p53, p53-target gene products, and reference proteins by immunoblotting, STK160830 down-regulated the expression of many of the proteins examined, and the downregulation correlated strongly with its inhibitory effect on cell death. mRNA expression analyses by qPCR and nascent RNA capture kit revealed that STK160830 showed a decreased mRNA expression, which was similar to that induced by the RNA synthesis inhibitor actinomycin D but differed to some extent. Furthermore, unlike other RNA synthesis inhibitors such as actinomycin D, p53 accumulation by STK160830 alone was negligible, and a DNA melting-curve analysis showed very weak DNA-intercalating activity, indicating that STK160830 is a useful inhibitor for RNA synthesis without triggering p53-mediated damage responses.
Highlights
Many biological responses are controlled by the up- or down-regulation of genes and gene products through transcription, translation, and post-translational modifications
Its inhibitory activity is based on inhibition of the kinase activity of P-TEFb [7,8,9], which phosphorylates the C-terminal domain of the largest subunit of RNA polymerase II, resulting in the induction of the p53 response [2]
It became clear that radiation cell death in thymocytes is p53-dependent, as thymocytes derived from p53 knockout mice were resistant to radiation-induced apoptosis [12,13]
Summary
Many biological responses are controlled by the up- or down-regulation of genes and gene products through transcription, translation, and post-translational modifications. These drugs revealed that radiationinduced thymocyte apoptosis requires de novo RNA synthesis and protein synthesis [10,11] After this discovery, it became clear that radiation cell death in thymocytes is p53-dependent, as thymocytes derived from p53 knockout mice were resistant to radiation-induced apoptosis [12,13]. It became clear that the previous conclusion that de novo RNA synthesis is not required for radiation cell death of MOLT-4 cells needs to be revised This finding could not have been found without the RNA synthesis inhibitory activity of STK160830, which scarcely induces a harmful p53 response. These findings indicate that STK160830 is an effective inhibitor to validate the requirement for transcription
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