Apoptosis

, Volume 16, Issue 9, pp 950–958

Elevated transcription of the p53 gene in early S-phase leads to a rapid DNA-damage response during S-phase of the cell cycle

Authors

  • Paula Takahashi
    • Department of Biological SciencesUniversity of South Carolina
    • Departamento de Genética, Faculdade de Medicina de Ribeirão PretoUniversidade de São Paulo
  • Amanda Polson
    • Department of Biological SciencesUniversity of South Carolina
    • Department of Biological SciencesUniversity of South Carolina
Original Paper

DOI: 10.1007/s10495-011-0623-z

Cite this article as:
Takahashi, P., Polson, A. & Reisman, D. Apoptosis (2011) 16: 950. doi:10.1007/s10495-011-0623-z

Abstract

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.

Keywords

p53DNA damageCheckpointsS-phaseApoptosis

Introduction

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 [1]. Consequently, it prevents accumulation of irreversible genetic alterations that could ultimately lead to cellular transformation [2]. These actions allow the maintenance of genome integrity throughout the cell cycle [3] 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 [8]. This rapid protein degradation is mediated by MDM2, whose transcription is activated by p53 itself [9]. 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 [13]. 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 [15].

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 [16]. 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 [18].

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, 1921]. 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 [2224]. 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 [25] 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 [26]. 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).

Chromatin immunoprecipitation

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×.

Results

p53 mRNA levels increase throughout the cell cycle

In order to examine p53 mRNA expression in S-phase in a synchronized cell population in the absence of DNA damage, Swiss3T3 cells were synchronized by serum depletion (0.1% FBS DMEM) for 24 h, followed by serum stimulation with 15% FBS DMEM for 3, 6, 9, 12, 13, 14, 16, and 18 h. The synchronization of the cell population was measured by 3H-Thymidine incorporation into DNA (Fig. 1a). RNA was extracted at each time point and subjected to RT-PCR analysis using p53 and GAPDH primers. p53 mRNA levels were quantified using ImageJ, an Image Processing and Analysis software that measures the pixel densities of each band in a selected area. Results were normalized to the 0 h time point (serum starvation for 24 h). The results demonstrate that in these synchronized cells, p53 mRNA levels decreased upon serum removal for 24 h (designated ‘0’ and indicates immediately prior to the addition of 15% serum), but are elevated early in S-phase (Fig. 1b). p53 mRNA expression begins to increase between 9 and 12 h post-serum stimulation as cells exit G1 and accumulates at maximal levels by 18 h after serum addition (Fig. 1b). By 24 h post serum treatment, the level of p53 mRNA returns to that seen in exponentially growing cells. Analogous findings have been published previously [21, 27, 28] and are consistent with other studies demonstrating that p53 mRNA synthesis in cells is enhanced during early S-phase when quiescent cells are induced to re-enter the cell cycle [6, 7, 1921].
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Fig. 1

a Exponentially growing Swiss3T3 cells (Exp) were serum-depleted for 24 h (0 h) and then serum stimulated. Cells were harvested at the indicated time-points. Cells were labeled with [3H]thymidine for 30 min followed by TCA precipitation and scintillation counting. The initial decline 3H incorporation in G1 is likely due to decreased repair synthesis. Experiments were performed in duplicate. b Quantification of p53 mRNA levels in S-phase in synchronized Swiss3T3 cells. Cells were serum-starved for 24 h (0) and subsequently serum-treated for 3, 6, 9, 12, 13, 14, 16, and 18 h. Extracted RNA was subjected to RT-PCR analysis using primers specific for p53 and GAPDH. mRNA levels were measured using ImageJ software and results were normalized to the 0 h time point. p53 mRNA level in exponentially growing cells was also measured and it indicated by the hatched line

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. [29] 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

After measuring mRNA levels of p53 and two of its targets and finding that p53 protein was not active in the absence of DNA damage, we assayed the rate of the p53 DNA damage response in synchronized cells entering S-phase. Activation of p53 was induced by treating cells with camptothecin, a DNA damage agent. Swiss3T3 cells were synchronized by serum depletion followed by serum stimulation with 15% FBS DMEM for 12 h. The 12 h time-point was assayed since this is the time that cells are beginning to enter S-phase (Fig. 1a) and the induction of p53 mRNA is detectable (Fig. 1b). At 12 h post-serum addition, camptothecin (5 μg/ml) was added and cells were harvested after 0 (no drug treatment), 10, 20, and 30 min, and 1, 2, 4, and 6 h post-drug exposure. Measurement of p53 mRNA levels indicated no additional increase upon treatment of the cells with camptothecin (data not shown) so cells were then treated as described above and western analysis was performed using anti-p53 monoclonal antibodies. To determine whether there was a difference in p53 levels between cells in early versus late S-phase, cells were incubated with 15% FBS DMEM for both 12 and 24 h prior to treatment with camptothecin. The results demonstrate that in both early and late S-phase the increase in the level of p53 protein was more rapid than in exponentially growing cells (Fig. 2). In the exponentially growing population, p53 protein levels were elevated between 4 and 6 h. With cells in S-phase, p53 protein expression was induced by camptothecin between 3 and 4 h in early S-phase and between 2 and 4 h in late S-phase (Fig. 2). Upon longer exposures of films to the western blots, increases in p53 were seen as early as 2 h after drug treatment (data not shown).
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Fig. 2

Western analysis of p53 protein in Swiss 3T3 cells that are exponentially growing or are in S-phase upon DNA damage. At 12 h post-serum addition, camptothecin (5 μg/ml) was added and cells were harvested at the indicated times post-drug exposure. 50 μg of protein were separated in 12% SDS-PAGE and transferred to Hybond-P nitrocellulose membrane. Membrane was probed with monoclonal anti-p53 and anti-actin antibodies

p53-mediated induction of Bax and p21 in response to DNA damage

To investigate the rate of the p53 DNA-damage response, we examined the expression of two p53 targets, Bax and p21. In order to measure the level of expression of these two p53 downstream targets after exposure to DNA damage during S-phase, we carried out RT-PCR analysis. These results showed that the Bax mRNA response to camptothecin treatment in non-synchronized cells was constant throughout the experiment (Fig. 3a). Both Bax and p21 mRNA levels were induced by 10- to 18-h post camptothecin treatment in exponentially growing cells (data not shown). Strikingly, however, the induction of Bax mRNA expression in synchronized cells in S-phase was rapid and remained higher (Fig. 3a).
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Fig. 3

Quantification of Bax and p21 mRNA levels in exponentially growing and synchronized Swiss3T3 cells in S-phase upon DNA damage. Cells were subjected to camptothecin treatment for indicated time points. Extracted RNA was subjected to RT-PCR analysis using primers specific for Bax (a) and p21 (b). mRNA levels were measured by densitometry and results were normalized to the exponential 0 h time point (no drug treatment). Results of two independent experiments were averaged

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.

To investigate the rate of increase in protein levels of these two p53 downstream genes upon camptothecin-induced DNA damage, Western analysis was carried out using monoclonal antibodies specific for Bax and p21. The results showed that Bax protein expression was induced by 4 h in exponentially growing cells, but by 1 h in cells in S-phase after DNA damage (Fig. 4a). Likewise, p21 protein expression started to elevate by 6 h after drug exposure in exponential cells, and by 4 h in synchronized cells both in early and late S-phase after drug treatment (Fig. 4b).
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Fig. 4

Western analysis of Bax and p21 proteins in exponentially growing and cells in S-phase upon DNA damage. 50 μg of protein were separated by PAGE and transferred to nitrocellulose. Membranes were probed with monoclonal anti-Bax (a), anti-p21 (b), and anti-actin antibodies. E exponential; S S-phase; C control (no drug treatment)

Binding of p53 to the Bax promoter in response to DNA damage

Chromatin immunoprecipitation (ChIP) analysis was performed in order to examine the rate of binding of p53 to the Bax promoter in response to DNA damage. Results of a series of ChIP assays are shown in Fig. 5 and demonstrate that cells not in S-phase and exposed to camptothecin, the binding of p53 to the Bax promoter remained constant throughout the experiment (Fig. 5). Conversely, in cells entering S-phase, an increase in p53 binding to the Bax promoter is observed after 0.5 h of drug exposure and continued to increase through the 4 h time-point. At the present time, the nature of the observed increased binding by p53 to the Bax promoter in serum-starved cells has not been characterized. It is likely, however, that without proper post-translational modifications that occur in response to DNA damage, the protein may in fact bind but not be competent to activate transcription [1, 2]. These results indicate that, in response to camptothecin treatment, Bax levels in cells entering S-phase are expressed in a more rapid manner than in cells that are not in S-phase. In response to DNA damage in cells in S-phase, p53 protein levels increase, bind to the Bax promoter, and cause a more rapid expression of this pro-apoptotic regulator (Fig. 3a).
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Fig. 5

ChIP analysis reveals p53 binding to the Bax promoter in response to DNA damage. Analysis of DNA/protein complexes formed between the Bax gene and p53 are measured by PCR using primers that were specific to the p53 binding site on Bax. The samples are represented by the “Input” DNA sample indicating total DNA present, No Antibody negative control, PBS negative control, normal mouse serum negative control, Exp No Camp, representing exponentially growing cells, and Exp 30 min, 1, 2, and 4 h indicating exponentially growing cells that were treated for the indicated time points with camptothecin. The Serum Starved sample underwent serum starvation for 24 h, but no camptothecin treatment. The S-phase cells were serum starved for 24 h, serum stimulated for 12 h to induce re-entry into the cell cycle, then treated with camptothecin for indicated time points

Rate of apoptosis in cells treated with camptothecin

After measuring the rate of response of p53, Bax, and p21 to DNA damage during S-phase, we assayed for induction of apoptosis after camptothecin treatment. The activity of caspases is an indicator of apoptosis. Therefore, the activities of two of these proteases, caspases 3 and 7, which are at the end of the apoptotic cascade, were measured. Exponentially growing and cells both in early and late S-phase were treated with camptothecin (as described above), harvested at 0 (no drug treatment), 3, 6, and 9 h post-drug addition, and luminescence (indicative of activity of caspases 3 and 7) of each experimental group was measured. The activity of caspases 3 and 7 increased between 3 and 6 h in exponential cells but between 0 and 3 h in cells both in early and late S-phase (Fig. 6). In addition to increasing more rapidly, the overall activity of these two caspases was higher in cells both in early and late S-phase (Fig. 6). These results indicate that apoptosis was induced earlier and to a greater extent in cells subjected to DNA damage during S-phase.
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Fig. 6

Activity of caspases 3 and 7 in response to camptothecin treatment in exponentially growing and cells in S-phase. Untreated (0) and camptothecin-treated (3, 6, and 9 h) exponentially growing cells in early and late S-phase were collected and subjected to Caspase-Glo 3/7 Assay. Luminescence was measured and normalized to 1000 cells. Results of two independent experiments were averaged

DNA fragmentation is also a marker of late stage apoptosis. Cells were treated with camptothecin for 18, 24, 36, and 48 h, harvested, and subjected to a TUNEL assay that labels the end of DNA fragments in cells undergoing apoptosis, ultimately allowing the staining of the nuclei of those cells. The results of these assays demonstrate that the number of cells with nuclei stained elevated with time after drug treatment for both populations, however, staining was more evident in cells in S-phase (Fig. 7). By 18 h after drug exposure, the number of cells in S-phase with fragmented DNA was greater compared to the same time point in non-S-phase cells (Fig. 6). These results support our conclusion that apoptosis is being initiated earlier and to a greater extent in cells that are subjected to DNA damage during S-phase.
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Fig. 7

Fragmentation of DNA (TUNEL) in response to DNA damage in exponentially growing cells and cells in S-phase. Untreated (control) and camptothecin-treated (18, 24, 36, and 48 h) cells were collected and subjected to TUNEL assay and pictures were taken using Zeiss Axioscope 40 at magnification 100×. Arrows point to examples of cells stained due to fragmented DNA

Discussion

Previous studies have demonstrated that p53 mRNA expression increases as normal cells enter S-phase [6, 7, 1921] 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 [30]. 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 [31]. 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. [12] 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 [32].

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. [33]. 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. [29] 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 [33], this would then insure a rapid and complete DNA damage response prior to the completion S-phase.

Acknowledgments

This work was supported by the Biomedical Research Infrastructure Networks (BRIN), and the South Carolina IDEA-Collaborative Research Program.

Conflict of interest

None.

Copyright information

© Springer Science+Business Media, LLC 2011