STL-based analysis of TRAIL-induced apoptosis challenges the notion of type I/type II cell line classification.
Bottom Line: Cells show distinct behaviors on several aspects of apoptosis, including (i) the relative order of caspases activation, (ii) the necessity of mitochondria outer membrane permeabilization (MOMP) for effector caspase activation, and (iii) the survival of cell lines overexpressing Bcl2.After having solved a few inconsistencies using STL-guided parameter search, we show that these three criteria do not define consistent cell line classifications in type I or type II, and suggest mutants that are predicted to exhibit ambivalent behaviors.Such tools are well-adapted to the ever-increasing availability of heterogeneous knowledge on complex signal transduction pathways.
Affiliation: INRIA Paris-Rocquencourt, Le Chesnay, France.
Extrinsic apoptosis is a programmed cell death triggered by external ligands, such as the TNF-related apoptosis inducing ligand (TRAIL). Depending on the cell line, the specific molecular mechanisms leading to cell death may significantly differ. Precise characterization of these differences is crucial for understanding and exploiting extrinsic apoptosis. Cells show distinct behaviors on several aspects of apoptosis, including (i) the relative order of caspases activation, (ii) the necessity of mitochondria outer membrane permeabilization (MOMP) for effector caspase activation, and (iii) the survival of cell lines overexpressing Bcl2. These differences are attributed to the activation of one of two pathways, leading to classification of cell lines into two groups: type I and type II. In this work we challenge this type I/type II cell line classification. We encode the three aforementioned distinguishing behaviors in a formal language, called signal temporal logic (STL), and use it to extensively test the validity of a previously-proposed model of TRAIL-induced apoptosis with respect to experimental observations made on different cell lines. After having solved a few inconsistencies using STL-guided parameter search, we show that these three criteria do not define consistent cell line classifications in type I or type II, and suggest mutants that are predicted to exhibit ambivalent behaviors. In particular, this finding sheds light on the role of a feedback loop between caspases, and reconciliates two apparently-conflicting views regarding the importance of either upstream or downstream processes for cell-type determination. More generally, our work suggests that these three distinguishing behaviors should be merely considered as type I/II features rather than cell-type defining criteria. On the methodological side, this work illustrates the biological relevance of STL-diagrams, STL population data, and STL-guided parameter search implemented in the tool Breach. Such tools are well-adapted to the ever-increasing availability of heterogeneous knowledge on complex signal transduction pathways.
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Mentions: For each initial protein concentration, one can predict the behavior of the system after TRAIL stimulation and assess whether this behavior satisfies a given STL property, or more precisely, estimate the value of the STL property given the behavior (see Methods). One can then graphically represent the value of the property in the state space by so-called phase diagrams (see Methods). The placement of cell lines in the phase diagram, based on their initial protein concentrations, indicates whether the cell line satisfies the given property (see Methods). Since it has been shown that the ratio of XIAP to caspase-3 concentrations plays a key role for the determination of the apoptotic type , we first constructed diagrams associated with these two variables. The corresponding STL phase diagram associated to Property 1 is represented in Figure 3. The death/survival property is tested in derived cell lines where Bcl2 is overexpressed (OE-Bcl2 cells; 10-fold increase of Bcl2 initial concentrations). The presence of two distinct regions in the diagram, one where Property 1 is satisfied (positive values, green) corresponding to cell survival, typical of type II cells, and one where Property 1 is falsified (negative values, red) corresponding to cell death, typical of type I cells, suggests that the model correctly predicts the importance of the XIAP/caspase-3 ratio as a key factor to determine cell survival following TRAIL treatment. We then positioned cell lines in the diagram based on measured mean and standard deviations of protein concentrations (see Methods). In agreement with the observations (Figure 2B in ) and the known type of these cell lines, the STL diagram predicts that OE-Bcl2 HCT116 cells do satisfy Property 1, but OE-Bcl2 SKW6.4 cells do not. OE-Bcl2 T47D cells are located close to separatrix and most cells satisfy Property 1. This is only in partial agreement with the fact that only half of T47D cells were found to survive (Figure 7C in ). Interestingly, as noted by Aldridge and colleagues, one can immediately see the consequences of mutations . For example, ΔXIAP cell lines are shifted to the leftmost part of the diagram (regions with low XIAP concentrations) and are thus predicted to violate Property 1. That is, all OE-Bcl2/ΔXIAP mutants of the HCT116, SKW6.4, and T47D cell lines are predicted to die in clonogenic experiments. This is again in accordance with experimental observations for HCT116 cells (Figure 2B in ). A detailed comparison of the Property 1 diagrams and the DLE diagrams used in  shows that the successful classification of cells provided by DLE diagrams implicitly relies on the snap-action, all-or-none aspect of apoptosis (Figure S2). Using the approach we propose here, the property of interest is explicitly stated and the interpretation of the resulting diagrams is not ambiguous. Moreover, since STL is a property specification language, this framework can be applied to analyze other properties of the system, not necessarily relying on snap-action responses.