Date of Award
Campus Access Thesis
The tumor suppressor p53 is commonly referred to as the "guardian of the genome" due to its ability to promote transcription of a variety of downstream targets that negatively regulate progression of the cell cycle in response to DNA damage or other cellular stressors, therefore, maintaining genome stability. Three of these targets are Bax (involved in mitochondrial apoptotic response), p21 (mostly involved in G1 arrest), and MDM2 (inhibitor of p53). Under stress conditions, p53 must be activated to avoid replication of cells containing damaged DNA. However, in healthy cells, when cell cycle arrest or apoptosis should not be induced, p53 must be inhibited. There are a number of studies that have demonstrated that transcription of p53 is activated prior to or during early S-phase in healthy cells. Because this is not what one would expect from a gene involved in growth arrest and apoptosis, we hypothesized that this is happening to provide mRNA (but no active protein) in order to prepare for DNA damage in S-phase, ensuring a rapid response before progression to the next stage of the cell cycle. We tested this hypothesis using synchronized Swiss3T3 murine fibroblasts. These cells were synchronized because in this way, more cells can be treated in S-phase, allowing one to see this rapid response to DNA damage. When comparing exponentially growing cells to synchronized cells upon DNA damage induced by camptothecin treatment, our results showed that p53 protein, but not mRNA, levels increased earlier in synchronized cells. Bax transcription was activated earlier and to a greater extent and protein was expressed earlier in synchronized cells as well. p21 transcription was activated to a greater extent in synchronized cells, but there was only a slight difference in protein expression. MDM2 transcription in turn, was induced to a lesser extent in synchronized cells. We also found that there was no difference in phosphorylation of serine 15 of p53 between exponential and synchronized cells. However, although phosphorylation of serine 392 of p53 happened at the same time in exponential and synchronized cells, there was a further increase in protein expression in both, and this further increase occurred earlier in synchronized cells. In addition, we reported that both activity of caspases 3 and 7, and DNA fragmentation occurred earlier and were stronger in synchronized cells. Therefore, our findings suggested that during S-phase of the cell cycle, the rate and extent of the p53 response to DNA damage is enhanced.
Takahashi, P.(2009). Increased Rate and Extent of the P53 Dna Damage Response During S-Phase of the Cell Cycle. (Master's thesis). Retrieved from http://scholarcommons.sc.edu/etd/99