p53 is dispensable for UV-induced cell cycle arrest at late G1 in mammalian cells

Mai A. Al-Mohanna, Fahad M. Al-Khodairy, Zbigniew Krezolek, Per-Anders Bertilsson, Khalid A. Al-Houssein and Abdelilah Aboussekhra,1

King Faisal Specialist Hospital and Research Center, Biological and Medical Research Department, MBC No. 03, PO Box 3354, Riyadh 11211, Saudi Arabia


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Genotoxic agents, including {gamma}-rays and UV light, induce transient arrest at different phases of the cell cycle. These arrests are required for efficient repair of DNA lesions, and employ several factors, including the product of the tumor suppressor gene p53 that plays a central role in the cellular response to DNA damage. p53 protein has a major function in the {gamma}-ray-induced cell cycle delay in G1 phase. However, it remains uncertain as to whether p53 is also involved in the UV-mediated G1 delay. This report provides evidence that p53 is not involved in UV-induced cellular growth arrest in late G1 phase. This has been demonstrated in HeLa cells synchronized at the G1/S border by aphidicolin, followed by UV exposure. Interestingly, the length of this p53-independent G1 arrest has been shown to be UV dose-dependent. Similar results were also obtained with other p53-deficient cell lines, including human promyelocytic leukemia HL-60 and mouse p53 knock-out cells. As expected, all of these cell lines were defective in {gamma}-ray-induced cell growth arrest at late G1. Moreover, it is shown that in addition to cell cycle arrest, HL-60 cells undergo apoptosis in G1 phase in response to UV light but not to {gamma}-rays. Together, these findings indicate that p53- compromised cells have a differential response following exposure to ionizing radiation or UV light.

Abbreviations: ATM, ataxia-telangiectaxia; HPV-16, papillomavirus 16; IR, ionizing radiation; MSF, mouse skin fibroblasts; UV, ultraviolet.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cells are constantly exposed to both endogenous and exogenous genotoxic agents that damage DNA and threaten their genomic stability (1). To efficiently repair damaged DNA and hence protect genetic information, growing cells of different eukaryotic species transiently arrest their growth at different points of the cell cycle (checkpoints). There are two major DNA damage-dependent cellular checkpoints, one at the G1–S transition before DNA replication (G1 checkpoint) and the other at the G2–M transition prior to mitosis (G2 checkpoint). The G1 checkpoint, which controls entry into S phase, prevents the replication of damaged DNA and its mutagenic consequences, and the G2 checkpoint prevents the segregation of aberrant chromosomes during M phase (2). In human cells, these delays play a major role in tumor suppression, since neoplastic cells from several forms of cancers were found to be defective in DNA damage-dependent cell cycle arrests (3). This response is under the control of a battery of genes which belong to different but overlapping pathways, underlying cellular growth delays in response to a wide variety of genotoxic agents, including ultraviolet (UV)-light and ionizing radiation (IR). Damaged DNA transmits an ill defined signal that enhances the transcription of some genes and functionally activates the products of others (2). The product of the tumor suppressor p53 gene is a major component of the cellular response to DNA damage in mammalian cells (4). DNA damage triggers accumulation and functional activation of p53, converting the protein from a latent to an active form. This seems to occur mainly through phosphorylation and acetylation processes which are triggered by different mediators, depending on the nature of the inducing signal. UV light and {gamma}-rays, for instance, which respectively induce cyclobutyl pyrimidine dimers and DNA strand breaks, use different signaling factors (5). The product of the gene inactivated in ataxia-telangiectasia (ATM) patients is a protein kinase responsible for the phosphorylation of p53 in response to ionizing radiation, but its role in response to UV light is limited. In fact, UV-damaged DNA mediates the signal to p53 through the ATR protein kinase (57). Moreover, the modification (8), stabilization (9) and kinetics of induction (10) of p53 in response to UV light or {gamma}-rays seem to differ significantly.

Once activated, p53 can direct the cellular machinery towards cell cycle delay and concomitant DNA repair and/or apoptosis, depending on the physiologic circumstances or cell type under study (1113). It is clear from several previous studies that p53 constitutes a major player in the cellular pathway activated in response to IR; however, its role in UV-dependent G1 arrest is still puzzling. As yet, no unequivocal evidence exists for p53 being required for UV-induced G1 arrest.

The work described here is an attempt to shed new light on the function of p53 in cellular response to UVC-induced DNA damage in the G1 phase of the cell cycle. To fulfil this, we first developed a synchronization protocol based on the combination of both serum starvation and aphidicolin treatment, which yielded a highly synchronized cell population. This allowed us to show that there exists a p53-independent G1 checkpoint for UV-induced damage in different p53-compromised cell lines, which are defective for {gamma}-ray induced G1 checkpoint. We also show here that G1-arrested HL-60 cells undergo apoptosis when treated with UV light but not in response to {gamma}-rays. These data indicate that in response to DNA injury, p53 protein plays different roles, depending on the nature of the genotoxic agent.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell lines
Human HeLa (cervical adenocarcima), Raji (Burkitt lymphoma), HL-60 (promyelocytic leukemia) and mouse MSF cells, p53+/+ and p53–/– derived, respectively, from B6129F2/J 101045 and B6129-Trp53tm1Tyj strains (14), were grown at 37°C with 5% CO2, in DMEM/Ham's F12 medium (50% each) supplemented with 15% fetal calf serum (FCS), 20 µg/ml gentamycin and 1% penicillin/streptomycin. HeLa and MSF cells were grown as monolayers, whereas Raji and HL-60 cells were grown in suspension.

Synchronization
HeLa or MSF cells (about 70% confluent) were seeded in T-60 plates and starved in serum-free medium for 24 h. Cells were then treated with 5 µg/ml aphidicolin in fresh medium supplemented with 15% FCS and incubated for another 24 h. For Raji and HL-60, 1x106 cells were plated in T-100 plates and treated like HeLa cells except that aphidicolin was present at a concentration of 2 µg/ml.

UV- and {gamma}-irradiation
UV-irradiation was delivered at room temperature using a germicidal UV light (chiefly 254 nm). The fluence rate of the UV light source, which was measured prior to each experiment with a UVX radiometer (Spectronics, NY) was about 1 J/m2/s. Cells were irradiated in the absence of medium, whereupon fresh medium supplemented with 15% FCS was added and finally the cells were immediately re-incubated at 37°C. {gamma}-Irradiation was administrated by a cobalt source at a dose rate of ~0.62 Gy/min.

Flow cytometry
Cell synchronization and cell cycle progression were monitored by flow cytometry. Approximately 1x106 cells from each sample were washed with PBS, harvested and fixed in 70% (v/v) ethanol. Cells were then treated with sodium citrate, RNase and stained with propidium iodide. Cell samples were analyzed on a FACScan flowcytometer (Becton Dickinson, San Jose, CA). Doublet discrimination mode was used to exclude doublets by means of gating PI fluorescence signal width (FL2-W) and area (FL2-A). CellQuest software (Becton Dickinson) was utilized for data acquisition and analyses.

Apoptosis analysis by DNA fragmentation assay
Cells were starved in serum-free medium for 24 h, then were irradiated either with UV light (10 J/m2) or {gamma}-rays (10 Gy) before replating them in the same starving medium. Cells were collected at various times, and DNA purified following the method used by Bissonnette and Hunting (15). In brief, 2x106 cells were harvested and incubated in 700 µl lysis buffer (10 mM EDTA and 0.6% SDS) and NaCl solution was added to give a final concentration of 1 M. After 18 h incubation at 4°C the lysates were centrifuged for 30 min. The supernatants were then incubated with proteinase K and brought to 1.3 M NaCl before isopropanol precipitation. Pellets were resuspended in TE and treated with RNase A. Finally, DNA was electrophoresed through a 1.2% agarose gel.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
HeLa cells are proficient in UV-induced late G1-checkpoint
HeLa cells are infected with papillomavirus 16 (HPV-16) which encodes the E6 protein. This oncoprotein functionally inactivates p53 by targeting it for rapid proteasome-mediated degradation via the ubiquitin pathway (16). Several studies have shown that the pathways both upstream and downstream of p53 are intact in cervical cancer (17). For these reasons, HeLa cells were used here to further explore the cellular response to UVC in the absence of functional p53. To accomplish this, it was essential to develop a synchronization protocol which yields highly synchronized cells, in order to study cellular behavior in response to DNA damage in a homogeneous cell population, which progress synchronously through the cell cycle. Starvation alone usually leads to the accumulation of cells in G0/G1 phase without allowing a good synchronization. To obtain a highly synchronous cell population, we combined both, starvation and treatment with aphidicolin, as outlined in Figure 1Go. Exponentially growing HeLa cells were starved for 24 h to arrest them in the G0/G1 phase of the cell cycle. This treatment did not increase the number of cells with G1 DNA content (Figure 1Go), and longer incubations (i.e. 48 and 72 h) under the same conditions did not improve the result (data not shown). Cells were then serum-stimulated by incubation for another 24 h in aphidicolin-containing medium. Aphidicolin is an inhibitor of DNA polymerases, which arrests cells at the G1/S border. Figure 1Go shows that following this treatment, more cells accumulated with a G1 DNA content; hence, only one peak at the 2n position is present (>70% of cells), while the second one representing cells with a 4n DNA content disappeared. This treatment allowed the accumulation of cells with G1 DNA content; however, the distribution of cells within G1 phase is not clear.



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Fig. 1. Synchronization of HeLa cells at G1/S border. Schematic representation of the synchronization strategy, illustrating the effect of the indicated treatments on cell cycle distribution.

 
To study the role of UV-induced DNA damage on cell cycle progression, aphidicolin was withdrawn and cells were divided into two sub-populations, one UV-irradiated (20 J/m2) and the other not irradiated to serve as a control. Cells were then allowed to resume cycling under normal growth conditions and collected at various times for flow cytometric analysis. Figure 2AGo shows that following the removal of aphidicolin, the control cells resumed cycling immediately in a synchronous manner, and 8 h later the majority (70%) had a S/G2 DNA content. This clearly shows that the cell population is greatly synchronized.



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Fig. 2. Cell cycle distribution of HeLa cells either following exposure to {gamma}-rays or UV light. Synchronized cells at G1/S boundary were either sham-irradiated or exposed to (A) UV, or (B) {gamma}-rays. Cells were immediately released to permit resumption of cycling and re-incubated for the period of time (h) indicated before being subjected to flow cytometric analysis. Results are representative of several experiments.

 
On the other hand, the vast majority of the UV-irradiated cells (60%) remained in G1 throughout the initial 12h subsequent to UV exposure with only a small fraction of cells (not more than 25%) entering S phase (Figure 2AGo). This shows that in contrast to the non-irradiated cells, the UV-treated ones were delayed at the G1/S border for ~12 h before resuming cycling. At 24 h post-irradiation, 32% of the UV-irradiated cells were in S phase, in comparison to only 12% of the non-irradiated cells. This observation reveals the existence of another UV-dependent delay in S phase. This parallels the facts that p53 is not required for triggering the DNA damage-induced S phase cell cycle checkpoint (1820). This result further indicates that HeLa cells are proficient in a prolonged cell cycle delay at late G1 in response to UVC.

Since p53 is a key player in cellular arrest upon exposure to IR, the response of HeLa cells to {gamma}-rays was analyzed to check whether or not they were deficient in delaying the cell cycle at late G1 phase following IR-damaging DNA. As for UV-irradiation experiments, cells were synchronized using the same protocol as described in Figure 1Go, and were then split into two sub-populations, whereupon one was exposed to {gamma}-rays (10 Gy) and the other acted as a non-irradiated control. Upon aphidicolin withdrawal, the irradiated and non-irradiated cells were released to resume cycling. Both subpopulations entered synchronously into S phase and had a significant G2 DNA content 10 h subsequent to the release, which was independent of the irradiation status (Figure 2BGo). However, as observed in response to UV at 24h post- irradiation, the {gamma}-ray-irradiated cells showed a delay in both S and G2 phases, not observed in the non-irradiated cells. Therefore, the S phase checkpoint seems normal in these cells when challenged by either UVC or IR. This indicates that most of the aphidicolin-treated cells were rather in late G1 than in early S phase. It is therefore clear that in contrast to UV light, {gamma}-rays did not induce growth arrest of HeLa cells at the G1/S border, implying that they are deficient in the G1 cell cycle checkpoint in response to {gamma}-rays but not to UV light.

What is the status of p53 in the G1/S arrested HeLa cells used in these experiments? Using an immunoblotting assay and G1-synchronized HeLa cells treated as described above, no p53 protein was detected either before or during the 24 h post-UV-irradiation. However, it was present in a Raji cell-free extract used as a positive control (data not shown). These results show that with respect to the G1 checkpoint, p53-compromised HeLa cells exhibit a differential response following exposure to ionizing radiation or UV light, suggesting that p53 is not essential for the latter.

UV-induced arrest at G1/S border is dose-dependent
To explore whether or not the UV-induced G1 arrest observed in HeLa cells could be obtained by lower UV doses, synchronized cells were split into four different populations. One was not irradiated (control) and the others were irradiated with three different UVC-fluences (5, 10 and 20 J/m2) and then released to reenter the cell cycle. Figure 3Go shows that the non-irradiated cells resumed cycling immediately after release with >60% of cells reaching S phase 4 h later. However, the UV-treated cells were delayed in G1. At 4 h post-irradiation with the three different doses, ~75% of cells were still in G1, indicating that the cellular growth of HeLa cells is delayed at the G1/S border even in response to very low UV fluence. Furthermore, the cell cycle delay period increased proportionally with UV dose, revealing a dose-dependent cellular growth arrest. This experiment showed a proficiency of the HeLa cells in the pathway sustaining cellular arrest in late G1 in response to UV light, despite the absence of functional p53 protein.



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Fig. 3. UV-induced G1 transient arrest in HeLa cells is dose-dependent. G1-synchronized cells were either sham-irradiated or treated with the indicated UV-fluences. Cells were then allowed to resume cycling and samples were collected at different intervals for flow cytometric analysis.

 
HL-60 cells exhibit UV-mediated late G1-arrest
To address whether other p53-deficient cells are also proficient in UV-induced G1 checkpoint control, we used the human promyelocytic leukemia HL-60 cell line which does not express p53 protein due to a large deletion in the gene (21). As for HeLa cells, HL-60 cells were synchronized in G1 by starvation, followed by aphidicolin treatment and finally sham-treatment or exposure to either UV (10 J/m2) or {gamma}-rays (10 Gy). Figure 4AGo shows that following aphidicolin treatment >70% of the HL-60 cells have a G1 DNA content. Six hours after the release, only ~29% of the non-irradiated cells remained with a G1 DNA content while ~50% entered S phase. At the same time, >55% of the UV-irradiated cells were delayed in G1 and only ~9% were in S phase (Figure 4bGo). Twenty-four hours subsequent to cellular release, the percentage of non-irradiated cells in S phase decreased to 14%, at which time ~30% of the irradiated cells had progressed to this phase. This finding shows that this cell line is still able to delay its cell cycle at G1/S border following UV treatment, and that this delay lasts for at least 8 h in response to 10 J/m2. As previously reported (22), HL-60 cells when treated with {gamma}-rays, did not show any delay of the cell cycle, and the evolution of the cell cycle was similar to that of the non-irradiated cells (data not shown). Similar results were obtained with the Raji cell line (wherein p53 is malfunctionally mutated) (data not shown), indicating that this cell line is also proficient in the UV-dependent G1 checkpoint. Therefore, in addition to HeLa cells, Raji and HL-60 cells exhibit a differential response following exposure to ionizing radiation or UV light, proving that p53, which is non-functional in all these cell lines, is not essential for UV-induced transient growth arrest in late G1 phase.





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Fig. 4. G1 delay and apoptosis in UV-treated HL-60 cells. G1-synchronized HL-60 cells were treated as described in Figures 1 and 2GoGo, except that the UV fluence was reduced to 10 J/m2. (A) Flow cytometric analysis. (B) Percentage of irradiated and non-irradiated cells in S phase. (C) Apoptotic cells in UV-irradiated and non-irradiated cell populations. The percentages in (B) and (C) were deducted from the results in (A).

 
UV-dependent induction of apoptosis in G1-synchronized HL-60 cells
The cell cycle delay displayed by HL-60 cells at the G1/S border was accompanied by an increased apoptotic response following UV exposure compared with non-irradiated cells (Figure 4AGo). The fraction of cells undergoing apoptosis was estimated by flow cytometry from the proportion of cells having a DNA content less than that present in the G0/G1 peak. Previous studies have shown that HL-60 cells die by apoptosis following DNA damage and that the sub-G0/G1 fraction correspond to apoptotic cells (2224). Analysis was carried out here on the same cells used to monitor cell cycle progression described above (Figure 4AGo). Figure 4CGo shows that apoptosis was observed very early after UV treatment and that the proportion of apoptotic cells (cells with less than G1 amount of DNA) increased proportionally with time. At 2 h post-UV-irradiation, only ~8% of cells were apoptotic. After 6 h of incubation, while the majority of UV-treated cells were still delayed in G1 phase (55%) (Figure 4AGo), ~28% underwent apoptosis. On the other hand, the fraction of cells that underwent apoptosis among the non-irradiated cells did not change significantly (Figure 4CGo). These results point to the ability of UV-treated HL-60 cells to concomitantly undergo both p53-independent apoptosis and cell cycle arrest. These cellular DNA damage responses have been rarely reported in the same cell type. These data also reveal that HL-60 cells are subject to UV-mediated apoptosis while they are in G1. To make sure that the proportion of apoptotic cells observed above does not correspond to the cells that escaped the cell cycle delay in G1, G1-synchronized cells were UV-irradiated and held in serum-free medium to keep them in G1. Even under these starving conditions, an increased apoptotic response was observed following UV treatment similar to that described in Figure 4CGo (data not shown). This suggests that UV-induced DNA damage triggers apoptosis in HL-60 cells even when the cells are in G1, in contrast to what was reported previously in response to {gamma}-rays (22). To test whether the cells used in the present study are also resistant to apoptosis when {gamma}-irradiated in G1 phase, HL-60 cells were starved in serum-free medium, treated either with UV or {gamma}-rays then re-incubated in the same starving medium. Cells were harvested at various intervals, and DNA was purified for the analysis of apoptotic DNA fragmentation. UV-treated cells underwent apoptosis, triggered as early as 4 h post-irradiation (Figure 5Go). However, IR-treated cells were resistant to apoptosis, which parallels what was previously reported (22). This shows that in G1 phase, the HL-60 cells display a differential apoptotic response following exposure to IR or UV light.



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Fig. 5. G1-arrested HL-60 cells undergo apoptosis in response to UV light but not to {gamma}-rays. Serum-free starved HL-60 cells were treated as shown and DNA was extracted at different post-irradiation times as described in Materials and methods. Lane C, non-starved cells; lane M, 100 bp DNA ladder.

 
p53–/– knock-out mouse skin fibroblasts are proficient in UV-induced cell cycle delay at G1/S border
After showing that p53 is not essential for UV-induced G1 arrest in different human tumor cell lines, we wanted to characterize this phenomenon further, making use of isogenic mouse skin fibroblast (MSF) strains. To do so, normal and p53–/– MSF cells were synchronized at the G1/S border as described above and were then either UV-irradiated (10 J/m2) or sham-treated. Next, cells were allowed to resume growth, and their cell cycle distribution was analyzed by flow cytometry. Figure 6AGo shows that in p53+/+ MSF cells, a delay in growth occurred following treatment with UV. After aphidicolin treatment, ~50% of the cells presented a G1 DNA content pattern, which did not significantly change 24 h subsequent to aphidicolin withdrawal (45% of cells still exhibited a G1 DNA content pattern). However, in the non-irradiated cells, this percentage decreased from 50% at the release time to 29% 24 h later, indicating a re-initiation of cellular growth in the non-irradiated cells. Figure 6BGo shows similar results, demonstrating that p53–/– MSF cells also underwent a cell cycle arrest following UV irradiation, while the non-irradiated cells resumed cycling. As in the case of the wild-type cells, the p53–/– cells were also arrested for up to 24 h, 64% of cells being in G0/G1 at the time of irradiation and the same distribution was found 24 h later. This shows that p53 is not required for cell cycle arrest at late G1 in mouse cells as well.




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Fig. 6. UV-induced cell cycle arrest in mouse skin fibroblasts. G1-synchronized MSF p53–/– (A) and p53+/+ (B) cells were either sham-treated or UV-irradiated with 10 J/m2 and the cell cycle distribution was measured by flow cytometry.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To cope with DNA damage, cells delay at different points in the cell cycle, activate specific DNA repair pathways and in some circumstances, induce programmed cell death (apoptosis). The product of the tumor suppressor gene p53, plays a central role in coordinating these responses in order to maintain genomic integrity and hence prevent neoplastic transformations. p53 is essential for the response underlying the cell cycle checkpoints triggered by {gamma}-rays; however, its role in the pathways leading to cell cycle delay in response to UV light awaits clarification.

In this report, we show that different p53-deficient human tumor cell lines (HeLa, HL-60 and Raji), in addition to mouse skin fibroblast cells, undergo UVC-induced cell cycle arrest at late G1 phase. This was achieved by developing a synchronization protocol, which allowed us to monitor the progression through the cell cycle of a high proportion of cells. This facilitated the comparison between the behavior of irradiated and non-irradiated cells. Cells were synchronized at G1/S border, and upon treatment with low UV-fluences, showed a delay in entering S phase as compared with non-irradiated cells. The duration of the growth arrest was dose-dependent, lasting for ~12 h in the case of HeLa cells treated with a UV-fluence of 20 J/m2. This could be explained either by a lack of p53 involvement in UV-induced G1 arrest in mammalian cells, or by the presence of two signal transduction pathways in response to UV-induced DNA damage, one p53-dependent and the second p53-independent. In the case of the cells used during the present study only the p53-independent pathway would be active. So far, there is no direct evidence that p53 is required for UV-induced G1 delay. It has been widely reported that human p53 protein accumulates and becomes activated in response to UV (10,25). This result only shows a UV-dependent p53 modification, but does not necessarily mean that UV light-induced G1 arrest is p53-dependent; in short the latter should be proven. However, there are indications that p53 is not part of the pathway mediating UV-induced G1 cell cycle arrest. The p53-compromised Li-Fraumeni fibroblasts, for instance, delay the cell cycle in G1 phase following UV irradiation (26). During these experiments, cells were accumulated in G0/G1 by starvation, then trypsinized and replated after irradiation. Moreover, it has also been shown that the p53 downstream effector, p21waf1, is transactivated in a p53-independent manner in both Li-Fraumeni fibroblasts and p53-knock-out mouse cells (26,27). Therefore, p21waf1 activation and its concomitant G1 arrest in response to UVC can take place independently of p53 through a hitherto undefined pathway.

On the other hand, p53 seems to be essential for the cellular network underlying the response to agents causing DNA strand breaks. In response to {gamma}-radiation, p21waf1 up-regulation and its concomitant cell cycle arrest are dependent on p53 (12,28). Moreover, the ATM protein is required for p53 activation and phosphorylation at its Ser15 residue in response to ionizing radiation (6,29). However, similar p53 phosphorylation occurs in response to UV-induced DNA damage (30), but ATM plays only a minor role in this response, which is under the control of the ATR protein kinase (6,7,31). These data show that p53 and ATM belong to the same pathway, specific for {gamma}-ray induced cell cycle checkpoint, but U-light dependent signaling is mediated through another pathway involving different effector components. Indeed, it has been shown recently that UV light induction of p53 correlated with inhibition of mRNA synthesis, which was, however, not significantly inhibited by the DNA strand-breaking agent ionizing radiation, although this agent causes cellular accumulation of both p53 and p21waf1 (25,32). Accordingly, UV and IR may trigger p53 induction/activation through different pathways, using different mediators and probably different DNA damage-sensing factors. Thus it would seem that there are different forms of activated p53, dependent on the nature of the agent triggering the p53 response. It was indeed recently shown that stabilization and activation of p53 are regulated independently by different phosphorylation events (33).

Since p53 does not seem to be required for the execution of the UV-induced G1 checkpoint, what could be the role of p53 phosphorylation/activation in response to UV-light? In addition to its role as a cell cycle checkpoint mediator, p53 is also involved in the nucleotide excision repair process (3440), apoptosis and the transcriptional activation of several genes (4,13). Hence, the activation of p53 by UV may be essential for one of these DNA metabolism processes such as DNA repair or apoptosis. In the present study we have shown that UV-treated HL-60 cells undergo p53-independent apoptosis as well as cell cycle arrest. Interestingly, apoptosis was observed following UV-treatment of G1-enriched population that had been starved after irradiation to hold the cells in the same phase. On the other hand, these cells were resistant to apoptosis when challenged with {gamma}-rays. These results show that UV but not {gamma}-rays can trigger apoptosis in HL-60 cells even when the cells are in G1, in the absence of DNA replication. This parallels what has been reported previously, concerning the cell cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumor agents. It has been shown that for some genotoxic agents, such as ionizing radiation and nitrogen mustard, apoptosis was cell cycle-phase specific. While IR preferentially triggers apoptosis in G2/M cells, nitrogen mustard preferentially activates apoptosis in G1 cells (22,41).

In conclusion, the response to UV light versus ionizing radiation seems to be regulated by different pathways in mammalian cells. Whereas p53 is essential for the response to {gamma}-rays, it has become clear that the protein has only a minor role in the UV-mediated G1 cell cycle checkpoint.


    Notes
 
1 To whom correspondence should be addressed Email: aboussekhra{at}kfshrc.edu.sa Back


    Acknowledgments
 
We wish to thank Drs M.A.Paterson, N.F.Dzimiri and M.A.Hannan for helpful discussions and critical reading of the manuscript. This work was supported by the King Faisal Specialist Hospital and Research Center, under the RAC proposal No. 990025.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 13, 2000; revised December 22, 2000; accepted January 4, 2001.