![]() ![]() To link the pharmacologic disruption of the p53-HDMX protein complex by SAH-p53-8 with reactivation of the p53 tumor suppressor pathway, we monitored the transcriptional activation of p53 targets by quantitative PCR (qPCR) analysis and the induction of apoptosis using a caspase-3/7 activation assay. Taken together, the immunoprecipitation and P-LISA data document that SAH-p53-8, but not Nutlin-3, inhibits the formation of p53-HDMX complexes due to its capacity to target intracellular HDMX. Furthermore, combining SAH-p53-8 with doxycycline/Nutlin-3 significantly reduced the P-LISA signal generated by the doxycycline/Nutlin-3 combination alone ( Figures 4D and 4E). Despite similar levels of p53 induction in response to single-agent SAH-p53-8 or Nutlin-3 treatment ( Figure 4C), the P-LISA signal generated by treatment with doxycycline was blocked upon cotreatment with SAH-p53-8 ( Figures 4D and 4E). The combination of doxycycline and Nutlin-3 treatment generated a robust P-LISA signal, which represents abundant formation of p53/HA-HDMX complexes ( Figures 4D and 4E). In the presence of doxycycline alone, the HA-HDMX produced binds to endogenous p53, leading to the formation of detectable but low-intensity P-LISA foci ( Figures 4D and 4E). ) were treated with doxycycline in the presence or absence of SAH-p53-8, Nutlin-3, or both. To confirm this observation in a cellular context, we employed a proximity ligation in situ assay, or P-LISA, and directly monitored p53-HDMX complex formation and pharmacologic dissociation in cells ( ![]() To stabilize p53 levels even further, we treated cells with the proteasome inhibitor MG-132 and correspondingly observed increased p53-HDMX complex, which was dose-responsively inhibited by SAH-p53-8 treatment ( Figure 4B). ![]() In contrast, p53-HDMX complexes were preserved, if not increased, in Nutlin-3-treated cells. ![]() Indeed, SAH-p53-8 effectively blocked the formation of p53-HDMX complexes ( Figure 4A). We examined whether SAH-p53-8 treatment prevents HDMX-mediated sequestration of p53, especially when p53 levels are further boosted by HDM2 blockade. Whereas an increase in p53 levels was observed upon treatment with either SAH-p53-8 or Nutlin-3 ( Figure 4A), only SAH-p53-8 treatment impacted JEG-3 cell viability ( Figure 3C). We found that JEG-3 cells had robust levels of p53 protein, which coimmunoprecipitated with HDMX ( Figure 4A ). After 6 hr treatment with vehicle, SAH-p53-8, or Nutlin-3, cellular extracts were prepared and subjected to anti-HDMX pull-down, followed by p53 western analysis. We performed immunoprecipitation studies in JEG-3 cells to interrogate whether the apparent pharmacologic advantage of SAH-p53-8 in Nutlin-3-resistant cells derives from HDMX targeting. This apparent discrepancy likely derives from (1) the preferential HDMX-binding activity of SAH-p53-8 compared to HDM2, thus lowering the effective concentration of SAH-p53-8 available for HDM2 targeting, and (2) the differential efficiencies of cellular import mechanisms for stapled peptides (i.e., pinocytosis Of note, despite the relatively enhanced capacity of SAH-p53-8 to displace the p53 transactivation helix from HDM2 compared to Nutlin-3 in vitro ( Figure 1C), Nutlin-3 was more cytotoxic than SAH-p53-8 in SJSA-1 cells ( Figure 3A). In contrast, SAH-p53-8 caused dose-dependent inhibition of cell viability in all five cell lines, suggesting that SAH-p53-8 is capable of reactivating the p53 pathway when cells express elevated levels of HDM2, HDMX, or both proteins ( Figures 3A–3E). MCF-7 and HCT116 cells were modestly sensitive to Nutlin-3 treatment, consistent with the coexpression of HDM2 and HDMX in these cells ( Figures 3D and 3E). Whereas SJSA-1 cells were very sensitive to treatment with Nutlin-3, SJSA-X and JEG-3 cells showed little to no response, consistent with the ability of Nutlin-3 to target HDM2 but not HDMX ( Figures 3A–3C). Cultured cells were treated with serial dilutions of Nutlin-3, SAH-p53-8, or the SAH-p53-8 F19A point mutant control. ![]()
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