Research Description

Manipulating Programmed Cell Death to Kill Cancer

Fluorescence approaches to understanding and manipulating apoptosis 
Ironically mitochondria both provide cells with the energy required to live and also serve as a platform that organizes programmed cell death or apoptosis (Kale et al., 2012). The complex signals that regulate the fate of the cell are integrated at the mitochondria and endoplasmic reticulum, and rely on the irreversible step of mitochondrial outer membrane permeabilization (MOMP) and/or an undefined signal from the endoplasmic reticulum that leads to loss of transmembrane potential at mitochondria. Apoptosis is normally used to get rid of unwanted cells during development, damaged cells in adults and prevents premalignant cells from progressing on to cancer. The same system governs selective kill of cancer cells by conventional chemotherapy agents. When the system fails or is inhibited, cancer frequently develops and cells become profoundly drug resistant.  As a process MOMP is hard to study because it is largely mechanical and occurs in membranes.  

Understanding Bcl-2 family proteins and how they function 
The Bcl-2 family of proteins plays a major role in both sensing different types of cellular stress and regulating MOMP. To accomplish these tasks, different members of the Bcl-2 family are located in multiple parts of the cell and function both as cytoplasmic and membrane proteins that adopt distinct conformations that dictate their function (Figure 1A).  The Bcl-2 proteins modulate apoptosis through a series of intricate protein-protein and protein-membrane interactions that exist in equilibrium.  We use and develop new fluorescence techniques to study the regulation of apoptosis in vitro and in cells. Fluorescence based assays uniquely permit quantitative analyses in real-time of the interaction mechanisms that govern the biology of complex systems.  By attaching fluorescent dyes to purified recombinant proteins we were able to reveal the ordering of the discrete steps in the activation of Bax by cBid in vitro (Figure 1B and Lovell et al, 2008). Fluorescence techniques can also be used to extend the results to live cells by expressing Bcl-2 family proteins fused with fluorescent proteins and measuring protein-protein interactions and binding dynamics by FRET (Figure 1C, Aranovich et al, 2012; Liu et al, 2012). We have used these techniques to identify novel small molecule inhibitors of Bcl-XL as well as Bax and Bak. These tool compounds are being used for a variety of studies including to validate new chemotherapy targets, to extend the life of stem cells, prevent damage during stroke etc. 

Personalizing medicine by evaluating cellular responses to genetic changes and drugs

Unlike apoptosis for many cellular processes we don’t know which proteins or genes are critically important and we don’t have detailed molecular assays that we can use to measure cellular responses the way we do for apoptosis.  For that reason we are developing new methods instruments and fluorescent dyes that can be used for both genetic and chemical screens in live cells.  Our main goal is determining how cells respond to drugs, particularly anti-cancer agents. However, we are applying these techniques to study a variety of cellular responses and to develop new approaches to personalized medicine. Using new dyes and image analysis techniques similar to those used for automated facial recognition from pictures we extract a large amount of data from fluorescence micrographs of cells.  We use this data to measure the biological effects of new candidate drugs on cell physiology or by knocking down the expression of genes we can validate new drug targets (Figure 2). We are currently using these techniques to examine cells from patients to optimize cancer treatment for individual patients.

Publications

View all publications on PubMed

BID preferentially activates BAK while BIM preferentially activates BAX, affecting chemotherapy response.
Sarosiek KA, Chi X , Bachman JA, Sims JJ, Montero J, Patel L, Flanagan A, Andrews DW , Sorger P, Letai A.
Mol Cell . 2013, 26:751-65.  Read

tBid Undergoes Multiple Conformational Changes at the Membrane Required for Bax Activation.
Shamas-Din A , Bindner S , Zhu W , Zaltsman Y , Campbell C , Gross A , Leber B , Andrews DW , Fradin C.
J. Biol. Chem . 2013, 288: 22111-27.  Read

Shedding light on apoptosis at subcellular membranes.
Kale J, Liu Q, Leber B, Andrews DW.
Cell. 2012, 151:1179-84. doi: 10.1016/j.cell.2012.11.013.  Read

Interactions of pro-apoptotic BH3 proteins with anti-apoptotic Bcl-2 family proteins measured in live MCF-7 cells using FLIM FRET.
Liu Q , Leber B, Andrews DW .
Cell Cycle . 2012, 11:3536-42. doi: 10.4161/cc.21462.  Read

Removing bias against membrane proteins in interaction networks.
Brito GC , Andrews DW.
BMC Syst Biol. 2011, 5:169. doi: 10.1186/1752-0509-5-169.  Read

Differences in the mechanisms of proapoptotic BH3 proteins binding to Bcl-XL and Bcl-2 quantified in live MCF-7 cells.
Aranovich A, Liu Q, Collins T, Geng F, Dixit S, Leber B, Andrews DW.
Mol Cell. 2012, 45:754-63. doi: 10.1016/j.molcel.2012.01.030.  Read