Abstract
Abstract Bcl-2 family members play a very important role in intrinsic apoptotic pathways. The family consists of more than 17 members that can be functionally separated into pro-apoptotic and anti-apoptotic subfamilies. Interactions between these family members determine cell fate, and dysregulation can lead to tumor growth and tumor cell survival. There are several investigational drugs for cancer treatment targeting Bcl-2 family members in clinical trials, driving the need for robust assays to measure Bcl-2 family members and their interactions. Though existing assays measure Bcl-2 family members, there is a lack of reliable assays to evaluate total proteins and protein-protein interactions simultaneously. To address this issue, we have developed two Luminex®-based multiplex immunoassay panels that allow for the simultaneous detection of multiple components of the Bcl-2 family in a single well, including Total Bcl-2, Bcl-xL, Mcl-1, BAD, BIM, and BAX as well as protein interactions like NOXA/Mcl-1, Mcl-1/BIM, and Bcl-xL/BAD. Using these panels, we analyzed changes in the expression and interactions of Bcl-2 family members in response to known apoptotic drugs, including Camptothecin (topoisomerase inhibitor), Anisomycin (protein translation inhibitor), and AT101 (BH3 mimetic drug). Camptothecin and Anisomycin each elicited a significant decrease in the levels of Mcl-1. Additionally, the Mcl-1/BIM interaction was disrupted by both Camptothecin and Anisomycin, perhaps a consequence of the reduced Mcl-1 levels. However, expression of the other anti-apoptotic proteins was unaffected by any of the three drugs. Interestingly, all three treatments had different effects on the Mcl-1/NOXA interaction. Mcl-1/NOXA signal was increased by AT101, decreased by Anisomycin, and showed a dose dependent effect with Camptothecin. In contrast to their disparate effects on the Mcl-1/NOXA interaction, all three drugs acted in a similar manner in blocking the Bcl-xL/BAD interaction without affecting the total levels of either protein. Overall, these results illustrate the dynamic responses of the Bcl-2 family to apoptosis-inducing drugs. The novel multiplex immunoassays described here provide a powerful tool for studying the underlying mechanisms regulating the Bcl-2 family of proteins. Citation Format: Reeti Maheshwari, Melissa Schluter, Danielle Pepin, Joseph Hwang. Analysis of Bcl-2 family members and protein-protein interactions using novel multiplex immunoassays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4319. doi:10.1158/1538-7445.AM2017-4319
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