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: It is important to note that the significantly different phenotypic responses of the different cell lines are in the model solely explained by observed differences in the initial concentrations of a dozen of key proteins. Therefore one can use EARM1.4 with new parameter values (Table S1) and STL diagrams to investigate the origins of the different behaviors shown by cell lines. One important question is to distinguish whether the different behaviors can be explained exclusively by differences in upstream protein concentrations or exclusively by differences in downstream protein concentrations, or whether a combination of upstream and downstream changes is needed . Indeed, it has been proposed that the main differences between type I and II behaviors are essentially due to differences in the efficiency of initiator caspase activation by the DISC , , . It has also been proposed that the main determinant is the concentration of XIAP relative to caspase-3 , . These questions can easily be answered using STL diagrams. Figure 8 shows the XIAP/caspase-3 and FLIP/caspase-8 diagrams for Property 2, computed with respect to the HCT116 cell line. It is apparent that the sole change of the concentrations of XIAP and capsase-3 from their original values to values corresponding to SKW6.4 cells is sufficient to alter the behavior of those cells from a type II to a type I phenotype. A similar change but for FLIP and caspase-8 proteins has no effect: cells remains with a type II phenotype. As illustrated in Figure S6 and S7, this is true for all properties and both directions (i.e., modifying protein concentrations from HCT116 to SKW6.4 values and vice-versa). This lack of influence of any upstream protein concentration is in apparent contradiction with the markedly different profiles for caspase-8 activation observed experimentally in  between type I and type II cells, and in EARM1.4 between HCT116 and SKW6.4 cell lines, and even more between normal and ΔXIAP cells that differ only in XIAP concentration (Figure 8C and 8D). The latter comparison suggests that differences in downstream protein concentrations feed back on upstream protein activities. To test this, we created feedback mutants (denoted ΔFB cells) by zeroing the cleaveage rate of caspase-8 by caspase-6. Similar activation profiles for caspase-8 in HCT116 and SKW6.4 cell lines were then obtained showing that in normal conditions differential activation of upstream processes is the consequence of differential downstream processes activation (Figure 8C and 8D). So, one can reconciliate the different views expressed by Scaffidi and colleagues and by Aldridge and colleagues , . There are indeed functionally significant differences in upstream protein activities (e.g. caspase-8) in type I and II cells. However, according to EARM1.4 model, these differences do not result from differences in upstream protein levels but rather from downstream differences that feed their influence back on upstream processes. The feedback loop is required to preserve synchronous initiator and effector caspase activation in type I cells. Note that using STL was instrumental here. Indeed, because all cells die in these simulations (no Bcl2 overexpression), DLE diagrams were not offering relevant information.