Elevated transcription of the p53 gene in early S-phase leads to a rapid DNA-damage response during S-phase of the cell cycle
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- Takahashi, P., Polson, A. & Reisman, D. Apoptosis (2011) 16: 950. doi:10.1007/s10495-011-0623-z
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p53 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. Since 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 sufficient p53 mRNA to ensure a rapid response to DNA damage before exiting S-phase. When comparing exponentially growing Swiss3T3 cells to those synchronized to enter S-phase simultaneously and treated with the DNA damaging agent camptothecin, we found that with cells in S-phase, 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.
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 . Consequently, it prevents accumulation of irreversible genetic alterations that could ultimately lead to cellular transformation . These actions allow the maintenance of genome integrity throughout the cell cycle  and because of that, p53 has been referred to as the “guardian of the genome” [4, 5].
Under normal conditions, p53 protein is unstable with a short half-life of 20–25 min [6, 7]. The very low levels of p53 in unstressed cells are a result of rapid protein turnover . This rapid protein degradation is mediated by MDM2, whose transcription is activated by p53 itself . MDM2 directly binds to the N-terminus of p53, where the transactivation domain is located, and inhibits its transcriptional activities. In addition to that, MDM2 is a ubiquitin E3 ligase that promotes p53 degradation by proteasomes [2, 10, 11].
Stress signals, including chemically induced DNA damage, X-rays, ultraviolet radiation, DNA synthesis inhibitors, hypoxia, and oncogene activation can trigger p53 activation. In response to those stimuli, p53 mRNA levels remain relatively constant; however, the protein half-life increases as a result of specific post-translational modifications [2, 12]. Once stabilized and activated, p53 accumulates in the nucleus and directly induces expression of a number of downstream target genes by binding to their promoters. Among those targets are the pro-apoptotic protein, Bax, and the regulator of cyclin/cyclin-dependent kinase (Cdk) complexes, p21 . Bax is a member of the Bcl-2 family of proteins and it is located in the cytoplasm. However, when apoptosis is induced, it moves to the outer mitochondrial membrane to promote permeabilization of the latter, therefore allowing leakage of, among other factors, cytochrome c from this organelle [11, 14]. These events lead to caspase cascade activation and ultimately apoptosis .
The activation of p21 by p53 regulates the transition between G1 and S-phase by interacting with and further repressing the kinase activity of the following Cdk complexes, cyclinD-Cdk4, cyclinE-Cdk2, and cyclinA-Cdk2 . In cells with unrepaired DNA that are already in S-phase, p21 binds to the complex composed of replication factor C, DNA polymerase δ, FEN1, and proliferating-cell nuclear antigen, causing their dissociation from the replication fork, which blocks synthesis of DNA [2, 16, 17]. p21 is also involved in G2 arrest by interacting with Cdc2, a Cdk that plays an important role in the progression into mitosis, and it does so by interacting with it .
Under normal conditions, when growth arrest or apoptosis should not be induced, p53 must be kept at low levels. Conversely, in the presence of stress signals, p53 expression must be induced to prevent accumulation of mutations, which could otherwise lead to genomic instability and carcinogenesis. Interestingly, however, a number of previous studies have found that p53 transcription is induced prior to or during early S-phase [6, 7, 19–21]. This increase in p53 transcription as cells enter S-phase in unstressed cells is notable since it is not necessarily predicted from a gene, such as p53, that induces cell cycle arrest and apoptosis. We hypothesized that p53 transcription occurs early in S-phase, so there will be available mRNA to quickly respond to DNA damage in S-phase. This would provide for a rapid response to DNA damage prior to the completion of S-phase. The response of p53 to DNA damage was measured during S-phase in synchronized versus exponentially growing Swiss3T3 cells. Cells in a synchronized population enter S-phase at approximately the same time and since there should be more p53 mRNA in early S-phase in a population of synchronized cells, we should detect a stronger and more rapid p53 response. In non-synchronized exponentially growing cells, the events of the S-phase response occur, but they occur at a relatively low level in the population as a whole and are more difficult to measure.
Levels of p53 mRNA and protein and two of its targets (Bax and p21) were assayed after inducing DNA damage with camptothecin, a drug that blocks DNA topoisomerase I catalysis and that halts synthesis of DNA and triggers DNA strand breaks [22–24]. Rates of entry into apoptosis in those cells were also analyzed by measuring the activity of caspases 3 and 7 and the extent of DNA fragmentation.
Materials and methods
Cell culture and camptothecin treatment
Swiss3T3 (murine fibroblasts with wild-type p53) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS, 100 U/ml penicillin, 100 μg/μl streptomycin, and 2 mM glutamine. Cells were cultured at 37°C in a humidified environment with 6% CO2. These cells were used for these experiments because they express wild-type p53, express many non-transformed characteristics such as contact inhibition, and have been shown to induce p53 expression upon DNA damage. Exponentially growing cells were 80% confluent when treated with 5 μg/ml of (S)-(+)-camptothecin (Sigma) for indicated times. Synchronized cells were grown exponentially until 80% confluent, serum-starved with DMEM containing 0.1% FBS for 24 h, serum-stimulated with DMEM containing 15% FBS for 12 h, and treated with camptothecin at 12 h post-serum stimulation for indicated times, unless otherwise specified.
DNA synthesis assay
3 × 105 Swiss3T3 cells were grown in DMEM with 10% FBS prior to serum depletion with DMEM containing 0.1% FBS for 24 h. Cells were stimulated with DMEM containing 15% FBS and harvested specified time-points. At each time-point, the medium was removed and replaced with medium containing 5 μCi/ml [3H]thymidine (70–90 Ci/mmol; Perkin-Elmer) for 30 min at 37°C. Radioactive thymidine was removed by washing with PBS containing 5 μg/ml non-radioactive thymidine. Samples were pelleted, lysed with 0.1 N NaOH to eliminate background due to inadvertent labeling of RNA  and precipitated onto 24 mm glass microfiber filter paper (Whatman) with trichloroacetic acid (TCA) and washed with ethanol. [3H]thymidine incorporation was assayed by scintillation counting.
RNA extraction and RT-PCR analysis
RNA was extracted and reverse transcribed (Ambion RETROscript). PCR reactions included 0.5 pmol/μl each forward and reverse primers, 0.1 pmol/μl each forward and reverse control primers, 0.025U/μl Taq polymerase, and 1 μl cDNA from reaction product. Amplifications with all primers consisted of 18 cycles and products were separated by electrophoresis. Pixel integrated densities were measured using ImageJ software using standard area for all bands. The sequences of primers used are as follows: murine p53 forward (5′-GGAAATTTGTATCCCGAGTATCTG-3′) and reverse (5′-GTCTTCCAGTGTGATGATGGTAA-3′) that result in a 183 bp product, murine Bax forward (5′-GCTCTGAACAGATCATGAAGACA-3′) and reverse (5′-CATGATG GTTCTGATCAGCTC-3′) that result in a 368 bp product, murine p21 forward (5′-TGTCCAATC CTGGTGATGTC-3′) and reverse (5′-TCTCTTGCAGAAGACCAATCTG-3′) that result in a 470 bp product . The sequences of control primers used are as follows: GAPDH forward (5′-AACGGATTTGGCCGTATTGG-3′) and reverse (5’′-CAGAAGGGGCGG AGATGATG-3′), β-actin/5 and β-actin/3 (Promega).
Protein expression and immunoblotting
Cells were lysed and sonicated and protein concentrations were measured by Bradford assays (BioRad). 50 μg protein were electrophoresed by SDS-PAGE and transferred to Hybond-P nitrocellulose membranes (GE Healthcare). Membranes were probed with the following mouse monoclonal antibodies: 53 Pab 421 (Calbiochem) and p53-DO-1 (Calbiochem), Bax (Ab-6, 6A7) (Calbiochem), p21 (Santa Cruz Biotechnology), and actin (Calbiochem) followed by anti-mouse Ig conjugated to horseradish peroxidase Detection was performed with ECL Plus (GE Healthcare).
Swiss 3T3 cells were grown to 80% confluence on three 15 cm plates (per experimental group). The media was removed and 20 ml of fixation solution (1.62 ml of 37% formaldehyde in 60 ml minimal cell culture media) was added to the cells for 30 min at 30°C and washed twice with PBS. The cells were resuspended in 1 ml lysis buffer, dounce homogenized, and sonicated. The sheared chromatin samples were stored at −70°C.
The cross-linked chromatin was precleared by adding blocked/washed Staph A cells for 15 min at 4°C. The samples were centrifuged and the supernatant was equally divided into 1.5 ml siliconized microcentrifuge tubes that represented ~1 × 107 cells for each immunoprecipitation. Approximately 1 μg each of p53 primary antibodies (DO-1 and Pab 421; Calbiochem, OP03L and OP43) was added to each of the samples. 2 μg of the nonspecific antibody, TIMP3, (Santa Cruz Biotechnology, Cat. No. K1103) were used as a nonspecific antibody negative control. The samples were incubated at 4°C overnight.
After immunoprecipitation and reversal of cross-linking the samples were PCR amplified using primers that amplified the p53 binding region on the Bax gene. The forward primer contained a sequence of 5′-GGGGCGCGCGGATCCATTCC-3′ and the reverse primer contained a sequence of 5′-GCTTCTGATGGACAGGGGGC-3′. For the “Nonspecific Primers” negative control, the forward primer contained a sequence of 5′-GCCCGTTGCCAGGCGCCGCCTTATAAA-3′ and the reverse primer contained the sequence 5′-GGCTCCAGGTAGGGGCTGAAGTCGA-3′. 5 μl of template DNA was used in each PCR reaction. The samples underwent 32 cycles of amplification.
Caspase-Glo 3/7 assay
Samples and Caspase-Glo 3/7 reagent were mixed in a 1:1 ratio and incubated at room temperature for 1 h. Luminescence (caspase 3/7 activity) was measured using a Zylux FB12 luminometer. The background (media plus reagent) was subtracted from the luminescence values (relative luminescence units), which were normalized to 1000 cells. Duplicates of two independent experiments were averaged.
DeadEnd colorimetric TUNEL assay
Cells were pipetted onto the poly-l-lysine-coated slides, allowed to air-dry and in 10% buffered formalin in PBS. Slides were washed with two changes of PBS and cells were permeabilized in 0.2% Triton X-100 solution. Slides were washed with PBS and labeling of fragmented DNA by rTdT was carried out using the DeadEnd Colorimetric TUNEL System (Promega). Slides were mounted in 100% glycerol and pictures were taken using Zeiss Axioscope 40 at magnification 100×.
p53 mRNA levels increase throughout the cell cycle
Since p53 mRNA expression increases in early S-phase in the absence of the addition of any DNA damaging agent, 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. Results showed that transcription of these two p53 downstream targets was not induced, thus, indicating that p53 protein is not active under these conditions. p53 protein levels as measured by western transfer remained undetectable (data not shown), however, the findings of Offer et al.  indicate that the level of non-active p53 protein appears to be elevated in early S-phase in murine pre-B cells.
p53 expression in response to DNA damage
p53-mediated induction of Bax and p21 in response to DNA damage
The p21 mRNA response to camptothecin treatment in both non-synchronized and synchronized cells in early S-phase increased after exposure to the drug, beginning by approximately 2 h post-drug exposure in non-synchronized cells but by 60 min post-drug exposure in early S-phase (Fig. 3b). These findings indicate that transcription of p53 target genes was being activated more rapidly and to a greater extent during the S-phase of the cell cycle.
Binding of p53 to the Bax promoter in response to DNA damage
Rate of apoptosis in cells treated with camptothecin
Previous studies have demonstrated that p53 mRNA expression increases as normal cells enter S-phase [6, 7, 19–21] and that this transcriptional regulation is due to the coordinated regulation by two transcription factors, C/EBP-β2 and RBP-Jκ, that bind to the p53 promoter [27, 28]. This is notable since p53 should not be present to induce growth arrest or apoptosis under normal conditions. One possible explanation for this rise in p53 mRNA levels would be that this provides a mechanism for rapid growth arrest or apoptosis in the event of DNA damage during S-phase. In order to test this notion, we measured the expression of p53, Bax, and p21 at the mRNA and protein levels as well as the rate of entry into apoptosis as cells enter S-phase.
Although levels of p53 mRNA are not further induced upon DNA damage in any of the cells tested, p53 protein was significantly induced in response to DNA damage (Fig. 2). These results are in agreement with the known findings that p53 activation is due primarily to post-transcriptional regulation . Bax transcription was induced more rapidly in cells in S-phase upon DNA damage (Fig. 3a). The observation that Bax appears to be induced before detectable levels of p53 protein is likely due to low levels of active p53 being able to induce Bax gene expression. In fact, upon longer exposures of films to the western blots, increases in p53 were seen as early as 2 h after drug treatment. Furthermore, it has been reported that Pea3 and Erm, two transcription factors that belong to the Ets family, appear to be involved in controlling the levels of Bax mRNA in normal mammary cells and that the adapter Yes-associated protein (YAP) increases transcription of Bax by p73 in H1299 human large cell lung carcinoma . Therefore, additional factors may also be participating in Bax expression in response to DNA damage.
It was also observed that p21 transcription was induced earlier and to a greater extent in cells in S-phase (Fig. 3b). The p21 response to DNA damage was markedly more rapid in late S-phase (Fig. 4b). This is consistent with the results of Kastan et al.  that demonstrated that upon DNA damage, while cells in G1 do not progress to S-phase, cells in S-phase only arrest in G2/M checkpoint, therefore, suggesting that p21 should accumulate later in S-phase and closer to the S/G2 transition rather than in early S-phase. It has also been reported that active p53, when highly expressed, usually induces permanent growth arrest in G1 and/or G2, and not S-phase .
The results presented here indicate that programmed cell death was induced earlier and to a greater extent during S-phase. This is consistent with recent findings published by Zhang et al. . These authors demonstrate that active E2F1, present during S-phase, cooperates with p53 to induce apoptosis rather than G1 arrest in response to DNA damage. The activities of the effector caspases 3 and 7 were higher and detected earlier during late compared to early S-phase in synchronized cells. This may be explained by the fact that at 12 h after serum stimulation, DNA synthesis is in an earlier stage, while by 24 h, DNA synthesis is complete. We speculate there is a stronger p53 response since in late S-phase there is more DNA being replicated compared to early S-phase. Thus, there is more DNA available to be damaged upon camptothecin treatment. Also, by late S-phase, it is likely that more p53 protein has accumulated in DNA damaged cells, leading to a stronger apoptotic response. These results are consistent with those reported by Offer et al.  who demonstrated the DNA repair activity occurs in a cell-cycle dependent manner and was enhanced in S- and G2 phases of the cell cycle. We conclude from these results that overall these findings support the hypothesis that p53 transcription takes place early in S-phase of the cell cycle, in order to provide sufficient p53 mRNA to enable cells to respond rapidly to DNA damage in S-phase. In cooperation with available E2F1 , this would then insure a rapid and complete DNA damage response prior to the completion S-phase.
This work was supported by the Biomedical Research Infrastructure Networks (BRIN), and the South Carolina IDEA-Collaborative Research Program.
Conflict of interest