DNA damage occurs during the course of our lives, spontaneously or through exposure to environmental mutagens. In response to DNA damage, the transcription factor p53 is activated. It then activates transcription of an array of downstream genes involved in cell arrest and repair, apoptosis and senescence. Not only is p53 involved in cancer avoidance, but it is also thought to play a role in aging. As the number of senescent cells or apoptotic incidents accumulates, multi-cellular organisms are thought to develop phenotypes associated with aging. How p53 mediates the transmission of the DNA damage signal into a decision to undergo repair, senescence or apoptosis is not well understood. This proposal aims to investigate whether the molecular dynamics of p53 play a role in the DNA damage response. P53 is regulated by a negative feedback loop;it is a transcriptional activator for its own negative regulator, Mdm2. As a result, p53 levels undergo oscillations for an extended period after DNA damage. This proposal hypothesizes that dynamic oscillations in p53 are not just a side effect of negative feedback, but that these dynamics play a physiological role in transmitting the DNA damage signal. Pulses of p53 followed by periods of absence may be beneficial to the organism because it keeps the incidence of apoptosis and senescence moderately low while still effectively minimizing cancer incidence. To test this hypothesis, p53 dynamic activity will be controlled experimentally, in the absence of Mdm2 feedback regulation. Experiments will go as follows: first DNA is damaged by exposing cells to gamma irradiation or a chemical mutagen. Then a computer controlled microfluidic device is used to add and take away a small molecule drug that controls the protein stability of an engineered p53 protein. This allows p53 dynamics, oscillations or constant expression, to be generated and evaluated. Transcriptional profiling of p53 target genes will determine whether oscillations play a role in the dynamics of the downstream effectors. Assays for senescence, apoptosis, genomic stability and other phenotypes associated with cellular aging will determine whether oscillations are a unique signal in determining cell fate. This project aims to be one of the first to demonstrate that oscillatory molecular behavior is beneficial because it allows the cell to "digitally" regulate a protein with toxic side effects.
This project seeks to determine whether oscillations in the level of the p53 protein play a unique role in deciding a cell's fate in response to DNA damage. Findings could suggest that some drugs (or drug activity) be administered in pulses to minimize unwanted side effects that cause premature aging.
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