Introduction

Eukaryotic cells are exposed continuously to the genotoxic stresses caused by various sources, such as  ionizing radiation (IR) and clastogenic drugs, but also may be formed endogenously during DNA replication or as an effect of reactive oxygen species (ROS). In order to maintain genomic integrity, the DNA damage response (DDR) is activated. DNA double strand breaks (DSBs) triggers series of events that determine cell fate. Incorrect mechanisms of DDR may lead to pathological changes transmitted to daughter cells, uncontrolled proliferation and tumor growth. The tumor suppressor p53 (cellular tumor antigen p53) is a main component of DDR pathway after DSBs induction, which increased production and activation may lead to various cellular responses to the damage: from cell cycle arrest and DNA repair processes to apoptosis. All of these complex mechanisms have one thing in common. In order to maintain the proper cellular response, the precise, fast and error-free damage detection is necessary with the proper signal transduction inside the cell.

For better understanding of the molecular mechanisms of the DDR, the useful approach is presented by systems biology.It treats the cell as a complex system of interactions between macromolecules (proteins, miRNAs, etc.) with multiple positive and negative feedback loops. The mathematical models approach allows not only to understand the complex relationship occurring inside the cell, but also allows to investigate and predict the effect of various stimuli, like stress agents, on the cell fate and cellular disorders (such as dysfunctional signal paths) that may lead to diseases of whole organism.

About the project

This project is a continuation of the research performed by its participants on developing mathematical model describing the dynamics of interactions occurring in p53 and NFκB signaling pathways activated after the formation of DNA damage (Puszyński2008, Puszyński2009). The authors developed a mathematical model of p53 and NFκB as a central module determining the cells fate, such as cell survival (proliferation) or apoptosis.

ATM protein kinase responds to the DSBs and is an activator of both pro-apoptotic signaling pathway of p53 and anti-apoptotic pathway of NFκB. Abnormalities that occur in these pathways may affect cell response leading to accumulation of mutations in the genetic material and development of different diseases, like cancer.

The authors developed a mathematical model of ATM regulatory pathway combined with p53-NFκB model. The parameters used for the equations from which the model is built were determined based on experimental data performed mostly by the team members. For the model development the authors used the Gillespie algorithm and its modification made ​​by Haseltine and Rawlings developed for numerical simulation of stochastic models (used forslow reactions, like states of genes change), andordinary differential equations (ODE) for the numerical simulation of deterministic models (used forquick reactions, like activation or degradation of the proteins).

The team works also on the ATR signaling pathway, which is responsible for detection of single stranded DNA and takes part in repair processes of DSBs. The existing model of p53 pathway (Puszyński2008) was connected to the ATR detector module. Now, the members of the team plan to connect the ATM-p53-NFκB with ATR detector system.

The importance of the project

The properly constructed mathematical model may allow to better understand the mechanisms that protect the cell from the consequences of inefficient DNA damage repair. Identification of potential abnormalities in the ATM/ATR-p53-NFκB pathway may lead to the development of new therapies for various human diseases. The model may be also used for preliminary analysis of the experimental hypotheses and putting the hypotheses on possible treatment at the level of the cellular signaling pathways, as well as lead to significant reduction in the cost of the experimental part of research and a more rational management of funds intended for biological experiments.

The main goal of the project is to develop a mathematical model describing the p53-NFκB regulatory pathway including ATM module as a detector system of DNA strand breaks. The model will allow to better understand the ATM-p53-NFκB signaling pathway and identify abnormalities that may occur during signal transduction through this pathway.

Project manager

Krzysztof Puszynski

Contractors

Krzysztof Puszynski, Patryk Janus, Monika Kurpas, Katarzyna Jonak

In the project the effects of ATM protein kinase on the p53-NF-kappaB pathway was studied. Moreover, the project included the investigation of main deactivation agent in his pathway - Wip1 phosphatase. 

Simulation analysis of the model show the typical relations known from literature and our biological experiments between Wip1 and proteins involved in the ATM-p53-NF-kappaB regulatory pathway:

• The silenced Wip1 to 25% affects the level of different proteins, like p53, Mdm2 or Chk2;
• With fully blocked transcription of Wip1 cells are more affected to apoptosis; 
• The absence of different components of the pathway in the model, like PTEN and Chk2, significantly affect the results of the simulation.

The results of the simulation analysis together with our experiments performed in the Institute of Oncology in Gliwice indicate that ATM is en effective system for DSBs detection with strong amplification signal and quick response. The authors noticed the strong dependence of the cellular response to the DNA damage on Wip1 and their regulators, like p53 and miRNA-16, what leads to the conclusion that it plays a role as a gatekeeper in the ATM-p53-NFkappaB pathway.

As a part of the project the simulation analysis of ATR-p53 pathway was performed. The model was activated by ultraviolet radiation (UV), what leads to formation of single stranded DNA. It was observed that:

• Changes of proteins levels during the simulation correspond to these described in the literature known to the authors;
• ATR module is able to detect even one single stranded break and enhance the signal to the p53 module;
• If the pathway is defective, the apoptotic threshold shifts. Despite the damages, the cell not die, but is able to transfer the incorrect genetic material to the next generations.