Cancer Modeling

The p53 tumour suppressor protein is a key regulator of the cell's response to geenotoxic stresses, including DNA damage, hypoxia, aberrant oncogenic activation and cellular ribonucleotide depletion. Because stress-induced p53 activation initiates a transcriptional program leading to cell cycle arrest, apoptosis or senescence, the p53 pathway is crucial for tumor prevention, as evidenced by the protein's mutation in over 50% of human tumors. A primary p53 target gene is its own negative regulator, Mdm2, whose gene product acts as an E3 ubiquitin ligase for p53. In unstressed cells, Mdm2 maintains p53 at very low levels, but following cellular stress this inhibition is relieved and p53 stabilizes, leading to oscillations of both p53 and Mdm2. Intriguingly, in vivo fluorescence measurements in individual cells revealed that in response to ionizing radiation (IR), p53 and Mdm2 exhibit a ‘‘digital’’ response that produces discrete pulses of p53 and Mdm2. The average height and duration of these pulses are fixed, whereas the mean number increases with the strength of DNA damage. While the biological significance of these oscillations remains unclear, they appear to involve a combination of post-translational modifications of p53, and the negative feedback loop consisting of p53 transactivation of Mdm2 and Mdm2-mediated ubiquitination of p53.

We have presented a model for the digital oscillatory p53 activity elicited by IR at the single-cell level. This model encompasses three processes: DNA damage and repair, modeled as a stochastic process; the p53-Mdm2 negative feedback oscillator, including a time delay representing tanscription, translation and transport; and an ATM switch that senses DNA damage levels and signals the downstream oscillator. Using this model, we have investigated the controlling role of ATM in setting a threshold level of DNA damage during the radiation response, as suggested by growing biochemical evidence. Moreover, by adding stochasticity to selected model parameters, we replicated the variable number of p53 pulses in individual cells as well as the cell population dynamics.

• L. Ma, J. Wagner, J.J. Rice, A. Levine, W. Hu and G. Stolovitzky. A plausible model for the digital response of p53 to DNA damage. Proceedings of the National Academy of Sciences, 102(40): 14266—14271, 2005. (PubMed)

p53–Mdm2 loop is controlled by a balance of its feedback strength and effective dampening using ATM and delayed feedback

Central to our p53 model is the existence of stable, limit cycle oscillations modulated by the DNA damage induced ATM signal. Moreover, our model is sufficiently parsimonious to be amenable to nonlinear systems analysis such as parameter variation studies and phase plane analysis. Generally speaking, biological systems are expected to have substantial variability, so demonstrating robustness against parameter variation becomes crucial for any model that is claimed to be biologically plausible. As a result, we have derived and analyzed a reduced, two-variable model that mimics qualitiatively our full p53-Mdm2 oscillator model. Analysis of this reduced model allowed us to examine explicitly how ATM modulates key components of the system, thus switching the response between fixed points and stable limit cycles. Moreover, comparison of this reduced model to a linear negative feedback model with time lag, whose stability condition we solve analytically, yielded a better understanding of how stability arises in time delayed negative feedback loops. Finally, we developed an interpretation of this linear model in terms of the strength of the feedback loop and the effective dampening of oscillations underlying negative feedback systems, and discuss ATM activation in this context.

• J. Wagner, L. Ma, J.J. Rice, W. Hu, A. Levine and G.A. Stolovitzky. p53–Mdm2 loop is controlled by a balance of its feedback strength and effective dampening using ATM and delayed feedback. IEE Journal of Systems Biology, 152(3):109—118, 2005. (PubMed)

A single nucleotide polymorphism in the MDM2 gene disrupts the oscillation of p53 and Mdm2 levels in cells

Our p53 model also predicted that specific ranges of both p53 and Mdm2 production rates were required to support oscillations following DNA damage. A recent report identified a single nucleotide polymorphism in the mdm2 promoter (SNP309), a T-to-G change at nucleotide 309 in the first intron of mdm2. This SNP results in an increased binding affinity of the transcriptional activator, Sp1, resulting in elevated Mdm2 expression and attenuated efficiency of the p53 pathway. In humans, SNP309 (G/G) is associated with an early age onset of, and increased risk for, tumorigenesis. Moreover, SNP309 (G/G) accelerates tumor formation in a gender specific (females) and hormone dependent manner. Consistent with these observations, we examined the possibility that high levels of Mdm2 protein in SNP309 (G/G) cells disrupted the regulation of p53 protein and the observerd oscillations, and investigated whether SNP309 affects the hormone dependent regulation of mdm2 transcription, as well as the oscillations.

To test the model’s predictions and further understand the impact of Mdm2 SNP309 on the function of the p53 pathway, the kinetics of production of p53 and Mdm2 proteins were analyzed after stress in various human cell lines with different SNP309 genotypes (T/T, T/G, G/G). Coordinated oscillations of p53 and Mdm2 proteins were observed in cells wild type for SNP309 (T/T) but not observed in cells homozygous for SNP309 (G/G) after IR. We also checked the impact of SNP309 upon the regulation of Mdm2 by estrogen. Estrogen preferentially stimulated the transcription of mdm2 from the SNP309 G allele and increased Mdm2 protein levels in estrogen responsive cell lines in a genotype-specific fashion. Regulation of basal p53 levels by tetracycline in H1299-HW24 cells demonstrated an intermediate range of p53 levels for which the oscillations were observed. Increasing p53 levels in the absence of IR did not generate oscillation, indicating that DNA damage is an important trigger of the p53-Mdm2 oscillation. These results suggest that disruption of the tight feedback loop of p53-Mdm2 may be an important reason to dampen the efficiency of the p53 pathway and increase susceptibility to cancer in individuals carrying SNP309 (G/G) in the Mdm2 gene.

• W. Hu, Z. Feng, L. Ma, J. Wagner, J.J. Rice, G. Stolovitzky and A. Levine. A single nucleotide polymorphism in the MDM2 gene disrupts the oscillation of p53 and Mdm2 levels in cells. Submitted.

Links to other persons and labs doing related work:

• Uri Alon's Laboratory
• Doug Green
• Michael Kastan
• Galit Lahav
• Andre Levchenko Laboratory
• Arnold Levine
• John Tyson's Laboratory