Abstract
The tumor suppressor gene TP53 is inactivated by mutation in a large fraction of human tumors. Around 10% of TP53 mutations are nonsense mutations that lead to premature termination of translation and expression of truncated unstable and non-functional p53 protein. Aminoglycosides G418 (geneticin) and gentamicin have been shown to induce translational readthrough and expression of full-length p53. However, aminoglycosides have severe side effects that limit their clinical use. Here, we show that combination treatment with a proteasome inhibitor or compounds that disrupt p53-Mdm2 binding can synergistically enhance levels of full-length p53 upon aminoglycoside-induced readthrough of R213X nonsense mutant p53. Full-length p53 expressed upon combination treatment is functionally active as assessed by upregulation of p53 target genes, suppression of cell growth, and induction of cell death. Thus, our results demonstrate that combination treatment with aminoglycosides and compounds that inhibit p53 degradation is synergistic and can provide significantly improved efficacy of readthrough when compared with aminoglycosides alone. This may have implications for future cancer therapy based on reactivation of nonsense mutant TP53.
Highlights
The tumor suppressor p53 (TP53) is a transcription factor that regulates a wide range of cellular processes including cell cycle progression, DNA repair, metabolism, apoptosis, and senescence [1, 2]
Induction of full-length p53 by G418 was accompanied by upregulation of p53 targets p21 and Wig-1 (ZMAT3) in a concentration-dependent manner, indicating that the recovered full-length p53 protein is at least partially functional as transcription factor (Figure 1B)
In order to further study if the full-length p53 protein induced by aminoglycosides in HDQ-P1 cells is transcriptionally active, we examined expression of p53 target genes by both Real-time PCR (RT-PCR) and Western blotting
Summary
The tumor suppressor p53 (TP53) is a transcription factor that regulates a wide range of cellular processes including cell cycle progression, DNA repair, metabolism, apoptosis, and senescence [1, 2]. P53 levels are low due to proteasomal degradation upon ubiquitination by the E3 ligase Mdm, encoded by the p53 target gene MDM2 [3]. In response to various forms of cellular stress, p53 accumulates and transactivates target genes such as CDKN1A (P21), GADD45, BAX, PUMA, NOXA, and TIGAR, leading to a p53-dependent biological response. One crucial function of p53 as tumor suppressor is the elimination of incipient tumor cells upon oncogenic stress [4]. The TP53 gene is mutated in a large fraction of human tumors [5, 6]. The majority of TP53 mutations (74%) are missense mutations that result in single amino acid substitutions in p53
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