Institute of Fundamental Technological Research

The p53 transcription factor is a regulator of key cellular processes including DNA repair, cell cycle arrest, and apoptosis. In this theoretical study, we investigate how the complex circuitry of the p53 network allows for stochastic yet unambiguous cell fate decision-making. The proposed Markov chain model consists of the regulatory core and two subordinated bistable modules responsible for cell cycle arrest and apoptosis. The regulatory core is controlled by two negative feedback loops (regulated by Mdm2 and Wip1) responsible for oscillations, and two antagonistic positive feedback loops (regulated by phosphatases Wip1 and PTEN) responsible for bistability. By means of bifurcation analysis of the deterministic approximation we capture the recurrent solutions (i.e., steady states and limit cycles) that delineate temporal responses of the stochastic system. Direct switching from the limit-cycle oscillations to the “apoptotic” steady state is enabled by the existence of a subcritical Neimark—Sacker bifurcation in which the limit cycle loses its stability by merging with an unstable invariant torus. Our analysis provides an explanation why cancer cell lines known to have vastly diverse expression levels of Wip1 and PTEN exhibit a broad spectrum of responses to DNA damage: from a fast transition to a high level of p53 killer (a p53 phosphoform which promotes commitment to apoptosis) in cells characterized by high PTEN and low Wip1 levels to long-lasting p53 level oscillations in cells having PTEN promoter methylated (as in, e.g., MCF-7 cell line).

Apoptosis, Cell cycle and cell division, DNA damage, DNA repair, Phosphorylation, Biochemical simulations, Cell cycle inhibitors, Transcription factors

Background
Apoptosis is a tightly regulated process: cellular survive-or-die decisions cannot be accidental and must be unambiguous. Since the suicide program may be initiated in response to numerous stress stimuli, signals transmitted through a number of checkpoints have to be eventually integrated.

Results
In order to analyze possible mechanisms of the integration of multiple pro-apoptotic signals, we constructed a simple model of the Bcl-2 family regulatory module. The module collects upstream signals and processes them into life-or-death decisions by employing interactions between proteins from three subgroups of the Bcl-2 family: pro-apoptotic multidomain effectors, pro-survival multidomain restrainers, and pro-apoptotic single domain BH3-only proteins. Although the model is based on ordinary differential equations (ODEs), it demonstrates that the Bcl-2 family module behaves akin to a Boolean logic gate of the type dependent on levels of BH3-only proteins (represented by Bad) and restrainers (represented by Bcl-xL). A low level of pro-apoptotic Bad or a high level of pro-survival Bcl-xL implies gate AND, which allows for the initiation of apoptosis only when two stress stimuli are simultaneously present: the rise of the p53 killer level and dephosphorylation of kinase Akt. In turn, a high level of Bad or a low level of Bcl-xL implies gate OR, for which any of these stimuli suffices for apoptosis.

Conclusions
Our study sheds light on possible signal integration mechanisms in cells, and spans a bridge between modeling approaches based on ODEs and on Boolean logic. In the proposed scheme, logic gates switching results from the change of relative abundances of interacting proteins in response to signals and involves system bistability. Consequently, the regulatory system may process two analogous inputs into a digital survive-or-die decision.

Apoptosis, Cell survival, Signaling pathway, Bcl-2 family, Bistability, Boolean logic, Ordinary differential equations

p53 is the key transcription factor controlling cellular responses to oncogenic stimulation and DNA da mage. Its activity is tightly controlled by numerous feedback loops. In response to DNA damage, p53 promotes expression of proteins, which suppress cell cycle and activate DNA repair. If the damage is irreparable or the repair takes too long, the programmed cell death (apoptosis) is initiated. In the current study we analyze the apoptotic module, a part of our larger p53 pathway model. In the model, the apoptosis is triggered due to the suppression of Akt activity and/or elevated level of p53 killer.p53 killer, i.e. p53 form phosphorylated at Ser-46 (in addition to Ser-15 and Ser-20), promotes synthesis of pro- apoptotic protein Bax. In healthy cells Bax is inactive due to binding to Bcl-2, another member of Bcl-2 family proteins. Suppression of Akt activity leads to the dissociation of Bax:Bcl-2 complexes and release of Bax. Therefore, two signals may lead to the accumulation of free Bax: one coming from elevated level of p53 killer, the other resulting from decreased level of active Akt. We demonstrated that, depending on parameters, the apoptosis can be controlled by the logic gate ‘AND’ as well as gate ‘OR’. In the first case both signals are required simultaneously, while in the latter case any of the two signals suffices for the initiation of apoptosis.