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Abstract
Abstractp53 induces the transcription of genes that negatively regulate progression of the cell cycle in response to DNA damage or other cellular stressors, and thus participates in maintaining genome stability. Under stress conditions, p53 must be activated to prohibit the replication of cells containing damaged DNA. However, in normal, non-stressed cells, p53 activity must be inhibited. Previous studies have demonstrated that p53 transcription is activated before or during early S-phase in cells progressing from G0/G1 into S-phase. In that this is not what would be predicted from a gene involved in growth arrest and apoptosis, in this study, we provide evidence that this induction occurs to provide available p53 mRNA in order to prepare the cell for DNA damage in S-phase, ensuring a rapid response to DNA damage before exiting this stage. When comparing exponential to synchronized Swiss3T3 cells upon Camptothecin-induced DNA damage, we found that in synchronized cells, p53 protein levels increased earlier, Bax and p21 transcription was activated earlier and to a greater extent and apoptosis occurred earlier and to a greater extent. These findings are consistent with p53 transcription being induced in S-phase to provide for a rapid DNA-damage response during S-phase of the cell cycle.
Highlights
The tumor suppressor p53 is a pivotal player in the negative regulation of cell cycle progression by promoting the transcription of numerous downstream regulators that either induce cell cycle arrest or apoptosis (Horn and Vousden, 2007)
Since we found that p53 mRNA expression increases in S-phase in the absence of DNA damage, in order to investigate if p53 protein was active, we performed RT-PCR analysis to measure mRNA levels of Bax and p21 under the same conditions
We found that p53 mRNA levels in both exponentially growing and synchronized cells did not change upon DNA damage throughout the experiment (Figure 2a). p53 mRNA expression in response to Camptothecin-induced DNA damage was similar in both exponentially growing and synchronized Swiss3T3 cells (Figure 2a)
Summary
The tumor suppressor p53 is a pivotal player in the negative regulation of cell cycle progression by promoting the transcription of numerous downstream regulators that either induce cell cycle arrest or apoptosis (Horn and Vousden, 2007). Synchronized cells showed a rapid increase in p53 binding to the promoter that peaks after 30min of drug exposure and remained high through the rest of the time course These results indicate that, in response to Camptothecin treatment, Bax levels in synchronized cells are expressed in a more rapid manner than in cells that are exponentially growing. Growing and synchronized cells (both in early and late S-phase) were treated with Camptothecin as described previously, harvested at 0 (no drug treatment), 3, 6, and 9h post-drug addition, and luminescence (indicative of activity of caspases 3 and 7) of each experimental group was measured. In addition to increasing more rapidly, the overall activity of these two caspases was stronger in synchronized cells both in early and late S-phase throughout the experiment (Figure 6) These results indicate that apoptosis was induced earlier and to a greater extent in synchronized cells. These results confirm our findings in the previous experiment that apoptosis is being initiated earlier and to a greater extent in synchronized cells
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