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Cell Growth & Differentiation Vol. 11, 149-156, March 2000
© 2000 American Association for Cancer Research

Role and Regulation of p53 during an Ultraviolet Radiation-induced G1 Cell Cycle Arrest1

Rory K. Geyer, Hatsumi Nagasawa, John B. Little and Carl G. Maki2

Harvard School of Public Health, Department of Cancer Cell Biology, Boston, Massachusetts 02115


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
p53 can play a key role in response to DNA damage by activating a G1 cell cycle arrest. However, the importance of p53 in the cell cycle response to UV radiation is unclear. In this study, we used normal and repair-deficient cells to examine the role and regulation of p53 in response to UV radiation. A dose-dependent G1 arrest was observed in normal and repair-deficient cells exposed to UV. Expression of HPV16-E6, or a dominant-negative p53 mutant that inactivates wild-type p53, caused cells to become resistant to this UV-induced G1 arrest. However, a G1 to S-phase delay was still observed after UV treatment of cells in which p53 was inactivated. These results indicate that UV can inhibit G1 to S-phase progression through p53-dependent and independent mechanisms. Cells deficient in the repair of UV-induced DNA damage were more susceptible to a G1 arrest after UV treatment than cells with normal repair capacity. Moreover, no G1 arrest was observed in cells that had completed DNA repair prior to monitoring their movement from G1 into S-phase. Finally, p53 was stabilized under conditions of a UV-induced G1 arrest and unstable when cells had completed DNA repair and progressed from G1 into S-phase. These results suggest that unrepaired DNA damage is the signal for the stabilization of p53, and a subsequent G1 phase cell cycle arrest in UV-irradiated cells.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The tumor suppressor protein p53 plays a critical role in the cellular response to DNA damage by functioning as a cell cycle checkpoint determinant (1) . Wild-type p53 levels are usually quite low because of a short protein half-life (2 , 3) . In contrast, p53 levels increase and the protein is stabilized in response to IR,3 and the cells undergo a G1-phase cell cycle arrest (2, 3, 4) . No G1 arrest is observed in IR-treated cells that lack p53, indicating an essential role for p53 in the arrest response (4, 5, 6) . The p53-dependent G1 arrest is thought to allow cells time to repair the damaged DNA before proceeding into S-phase, thereby preventing an accumulation of mutations that could occur from replicating a damaged genome. Consistent with this hypothesis are reports that loss or inactivation of p53 causes cells to accumulate mutations at a higher rate (7 , 8) . p53 can also trigger apoptosis (programmed cell death) in certain cell types after irradiation treatment (9 , 10) . For example, thymocytes from p53 knockout mice were more susceptible to radiation-induced apoptosis than were thymocytes from cells expressing p53 (9) . On the basis of these results and others, it has been proposed that the normal function of p53 is to monitor the integrity of the genome and protect cells from accumulating genetic damage. p53 carries out this function by temporarily halting cell proliferation to allow DNA repair or by eliminating DNA damaged cells through apoptosis.

In contrast to IR, a role for p53 in response to UV radiation has not been clarified. p53 levels increase in UV-irradiated cells as they do after IR treatment, and the cells undergo a G1 arrest. However, in some cases this G1 arrest was observed in normal cells and in cells in which p53 was inactivated by expression of either SV40 large T-antigen or the E6 oncoprotein of human papillomavirus (11 , 12) . These results suggested that the UV-induced G1 arrest occurs in a p53-independent fashion. In contrast, a moderate G1 arrest that appeared to be p53 dependent was observed recently in cells exposed to low doses of UV radiation (12) . Furthermore, a transient G1 arrest was observed in UV-irradiated human oral keratinocytes that expressed wild-type p53 but not in keratinocytes that lacked wild-type p53 expression (13) . These results suggest that, at least in some cases, p53 can play a role in the establishment of a G1 arrest after UV radiation treatment.

It has also been suggested that p53 may play a direct role in DNA repair after UV radiation treatment. UV radiation causes pyrimidine dimer formation and generates (6-4) photoproducts in DNA, both of which are repaired through a process called NER (14) . Expression of wild-type p53 was reported to be necessary for efficient NER in UV-irradiated human fibroblasts, suggesting that p53 may play a role in the NER process (15) . The best characterized NER components are the XP factors, of which there are seven, designated XP-A to XP-G. XP-B and XP-D are DNA helicases and critical components of the NER pathway (16 , 17) . p53 can interact directly with XP-B and XP-D and inhibit their helicase activities in vitro (18) . These results raise the possibility that p53 may function during NER by modulating the activities of these two helicases. In contrast, Wang et al. (19) reported that XP-B and XP-D are required components of a p53-mediated apoptosis pathway (19) . Therefore, the interaction between p53 and either XP-B or XP-D may mediate an apoptotic function of p53, without affecting DNA repair.

Given the role of p53 in cell cycle control and its potential role in NER, it is important to determine the relationship between UV radiation, p53, and DNA repair. In this study, we used normal and repair-deficient cell lines to examine the role and regulation of p53 in response to UV radiation. A dose-dependent G1 cell cycle arrest was observed in normal and repair-deficient cells exposed to UV. Expression of HPV-16 E6, or a dominant-negative p53 mutant that inactivates wild-type p53, caused cells to become resistant to this UV-induced G1 arrest. However, cells in which p53 was inactivated still underwent a significant G1 to S-phase delay after UV exposure. These results indicate that UV can inhibit G1 to S-phase progression through p53-dependent and independent mechanisms. Repair-deficient cells were more prone to a UV-induced G1 arrest than normal cells. Furthermore, no G1 arrest was observed in normal cells that had completed DNA repair prior to monitoring their movement from G1 into S-phase. Finally, p53 was stabilized under conditions of a UV-induced G1 arrest and unstable when cells had completed DNA repair and progressed from G1 into S-phase. These results suggest that unrepaired DNA damage is the signal for the stabilization of p53 and the subsequent p53-dependent G1 arrest in UV-irradiated cells.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Role of p53 in a UV-induced G1 Block or Delay.
The purpose of this study was to examine the role and regulation of p53 during the cell cycle response to UV radiation. Toward this end, normal human fibroblasts (GM6419 cells) were infected with control retroviruses or retroviruses that express the HPV-16 E6 oncoprotein. HPV-16 E6 promotes the rapid degradation of p53 through the ubiquitin-proteolysis pathway (20, 21, 22) , and cells that express E6 are therefore similar to cells that lack p53. The effect of UV radiation on the progression of these cells from G1 into S-phase was then assessed. Cells were maintained at confluence for 48 h to obtain G1-phase cell populations. The cells were then treated with increasing doses of UV radiation and replated at low density to stimulate there movement from G1 to S-phase. Progression from G1 into S-phase was monitored by FACS analysis. As shown in Fig. 1, A and B,Citation >90% of the cells had a G1 DNA content at the zero time point. The percentage of nonirradiated G1 phase cells decreased between 12 and 18 h after growth stimulation because of the movement of cells into S-phase. A UV dose of 4 J/m2 caused a delay in the movement of control virus-infected cells into S-phase, and a UV dose of 8 J/m2 caused a complete G1 arrest up to 42 h after irradiation. Eight J/m2 appeared to be the minimum dose that could cause a complete G1 arrest in control virus-infected GM6419 cells (not shown). Cells expressing HPV-16 E6 were resistant to a UV-induced delay at 4 J/m2, and their movement into S-phase was delayed, although not completely inhibited, at a UV dose of 8 J/m2. p53 and p21 protein levels were also monitored in the nonirradiated and irradiated cells (Fig. 1C)Citation . In control virus-infected cells that were not irradiated, p53 and p21 levels were unchanged or slightly decreased after growth stimulation. In contrast, p53 and p21 levels were increased in cells treated with 8 J/m2 prior to plating and growth stimulation. Furthermore, p53 and p21 levels were low in cells expressing HPV-16 E6, and neither p53 nor p21 were induced upon UV treatment. These results are consistent with the UV-induced arrest resulting, at least in part, from activation of the p53-p21 growth arrest pathway.



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Fig. 1. GM6419 cells that were infected with a retrovirus expressing HPV-16 E6 or a control retrovirus (LXSN) were maintained at confluence for 48 h. The cells were then UV irradiated (0, 4, or 8 J/m2) and plated at low density to stimulate their movement from G1 into S-phase. Cell cycle distribution was determined by FACS analysis at various time points after plating. A, representative FACS data from a single experiment is illustrated and shows a complete G1 arrest in control virus-infected cells exposed to 8 J/m2 and a G1 to S-phase delay in E6-expressing cells exposed to 8 J/m2. B, the percentage (%) of cells with a G1 DNA content at each time point from an experiment similar to that in A is plotted. The decrease in the percentage of G1 is attributable to the movement of cells from G1 into S-phase. C, cells were either nonirradiated or exposed to UV (8 J/m2) and plated as described above. At the indicated time points after plating, protein extracts were prepared. One hundred µg of each extract were examined by Western blot analysis with the p53 antibody Ab-6 (Oncogene Science) or the p21 antibody 15431E (PharMingen).

 
The ability of E6 to overcome a UV-induced G1 arrest could have resulted from inactivation of p53 or from other E6 activities. To confirm the involvement of p53 in this UV-induced G1 arrest, GM6419 cells were infected with a retrovirus encoding a dominant-negative p53 mutant (p53-CTF) capable of inactivating the wild-type p53 protein (10) . The effect of UV radiation on the progression of these cells from G1 into S-phase was then assessed (Fig. 2A)Citation . As with E6 expression, cells that expressed p53-CTF were resistant to a UV-induced G1 phase arrest after exposure with 8 J/m2 (Fig. 2A)Citation . These results indicate that inactivation of p53 by either the dominant-negative p53 mutant or HPV-16 E6 can overcome a UV-induced G1 arrest. A G1 to S-phase delay was still observed after exposure to 8 J/m2 in p53-CTF-expressing cells, indicating that UV can also induce a G1 to S-phase delay that is independent of p53. Steady-state levels of p53 were increased in cells expressing p53-CTF, attributable to the fact that the p53-CTF mutant can stabilize the endogenous p53 protein by sequestering it in inactive complexes (10) . Nonetheless, levels of full-length p53 and p53-CTF were unchanged after UV treatment of the p53-CTF-expressing cells, and p21 protein levels were undetectable even after UV exposure (Fig. 2B)Citation . It should also be noted that p21 as well as MDM2 protein levels were low and not increased after IR treatment of the p53-CTF-expressing cells (not shown). Taken together, these results indicate that the p53-CTF mutant functionally inactivated the endogenous p53 protein.



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Fig. 2. A, GM6419 cells that were infected with a retrovirus expressing a dominant-negative mutant form of p53 (p53-CTF) were grown to confluence to obtain G1-phase cells. The cells were then untreated or exposed to a UV dose of 8 J/m2 and plated at low density to stimulate their movement from G1 to S-phase. Cell cycle distribution was determined by FACS analysis at various time points after plating. The percentage of cells with a G1 DNA content at each time point is indicated. B, cells were either nonirradiated or exposed to UV (8 J/m2) and plated as described above. At the indicated time points after plating, protein extracts were prepared. Thirty µg of each extract were examined by immunoblotting using the p53 antibody Ab-6 for full-length p53 or the p53 antibody Ab-1 for p53-CTF, and 100 µg of extract were examined by Western blotting using the p21 antibody 15431E. The positive control for the p21 blot was 100 µg of extract from control retrovirus-infected GM6419 cells treated with 8 J/m2 UV and harvested 21 h after plating.

 
p53 Mediates a UV-induced G1 Block in UV Repair-deficient Cells.
G1 to S-phase progression was delayed in GM6419 cells exposed to 4 J/m2 and completely blocked at a UV dose of 8 J/m2, indicating that the extent of G1 arrest after UV treatment was dose dependent. We predicted, based on these results, that cells deficient in the repair of UV-induced DNA damage would be more susceptible to a UV-induced G1 arrest than normal cells. Patients with XP cannot efficiently repair UV-induced DNA damage (14 , 23) . XP cells from complementation group C (XPC cells) repair damage to actively transcribed DNA strands normally but are defective in the repair of nontranscribed DNA regions (24) . XP cells from complementation group D (XPD cells) are defective in the repair actively transcribed DNA regions (25) . XPC and XPD cells were infected with control retroviruses or retroviruses that express HPV-16 E6 or p53-CTF, and the effect of UV on their movement from G1 to S-phase was assessed. Immunoblot analyses similar to that shown in Fig. 2Citation demonstrated p53-CTF expression in the XPC and XPD cells infected with the p53-CTF-expressing retrovirus (not shown). The minimum dose that caused a complete G1 arrest up to 60 h after irradiation was ~1.5 J/m2 in the XPD cells and 5–6 J/m2 in XPC cells (Fig. 3)Citation . It is important to note that similar results were obtained with one other XPC and XPD cell line (not shown). Expression of either HPV-16 E6 or p53-CTF abolished the UV-induced G1 arrest in these repair-deficient cells, indicating that the arrest was mediated in part by p53 (Fig. 3A)Citation . As in GM6419 cells, inactivation of p53 in these repair-deficient cells did not completely overcome the effects of UV, because a UV-induced G1 to S-phase delay was still observed in cells expressing HPV-16 E6 or p53-CTF. Immunoblot analyses (Fig. 3B)Citation indicated that p53 and p21 levels were induced by UV radiation in control cells but not induced in cells infected with either the HPV-16 E6 or p53-CTF retroviruses, consistent with the UV-induced G1 arrest resulting in part from activation of the p53-p21 growth arrest pathway.



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Fig. 3. A, XPC and XPD cells that were either uninfected or infected with a retrovirus expressing HPV-16 E6 or the dominant-negative mutant form of p53 (p53-CTF) were grown to confluence to obtain G1-phase cells. The cells were then either untreated or exposed to the indicated UV dose, followed by plating at low density. Cell cycle distribution was determined by FACS analysis at various time points after plating. The percentage of cells with a G1 DNA content at each time point is indicated. B, cells were either untreated or exposed to UV and plated as described above. At the indicated time points after plating, protein extracts were prepared and examined by Western blotting for p53 and p21. Thirty µg of protein extract from cells expressing the dominant-negative p53 mutant was loaded in each lane for the p53 Western blot. In all other cases, 100 µg of protein were loaded per lane. The positive control (+ ctrl.) for the p21 blot was 100 µg of extract from noninfected XPD or XPC cells treated with UV and harvested 21 h after plating.

 
Unrepaired DNA Damage Mediates a UV-induced G1-Phase Block.
Because the repair-deficient cells were more susceptible to a UV-induced arrest than normal cells, we suspected that unrepaired DNA damage may be the signal for a UV-induced arrest. To examine this possibility, cell cycle progression was analyzed in UV-irradiated cells that were first allowed to repair their DNA before being stimulated to move from G1 into S-phase. DNA repair activity (UDS) was assessed in UV-irradiated GM6419 cells as described previously (26) . Briefly, G1 phase cells were UV irradiated and maintained in G1 for 24 h. At various time points after UV treatment, the cells were pulse labeled with [3 H]thymidine. Because the cells were in G1, the uptake of [3 H]thymidine was attributable to DNA repair synthesis only and not because of replicative DNA synthesis. The uptake of radionucleotide at each time point was monitored by fixing the cells directly to the culture dish and exposing them to a photographic emulsion prior to autoradiographic development. The average number of silver grains precipitated from the emulsion per cell nucleus was determined by microscopic examination and is a measure of DNA repair activity (UDS). The data are plotted in Fig. 4Citation as % UDS at various time points after UV treatment. The level of UDS was maximal immediately after UV treatment (100% UDS) and diminished to background levels after 24 h of holding in G1 (Fig. 4A)Citation . These results indicate that DNA repair after UV treatment was completed during the 24-h period that the cells were held in the G1 phase.



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Fig. 4. Upper panel, GM6419 cells were maintained at confluence in 35-mm tissue culture dishes to obtain G1-phase cells. The cells were then UV irradiated and maintained in the G1 phase. At 4, 8, or 24 h after UV treatment, individual plates were incubated in the presence of 1 mCi/ml [3H]thymidine for 1 h. The cells were fixed to the plate and exposed to a photographic emulsion for 1 week and then processed by autoradiography. The number of silver grains precipitated from the emulsion per cell nuclei was counted by microscopic examination and was used as a measure of repair DNA synthesis (UDS). The experiment was done in duplicate, and a minimum of 50 cells were examined on each individual plate. The highest level of repair DNA synthesis was observed immediately after UV treatment (0 time point) and was considered 100% UDS. UDS was completed after 24 h holding in G1. Lower panel, G1-phase GM6419 and GM2996 (XPC) cells were either untreated or exposed to UV doses of 8 or 6 J/m2 as indicated. The cells were then plated at low density to stimulate their movement from G1 to S-phase, and cell cycle distribution was determined by FACS analysis. Cells were either plated immediately after UV treatment or were held at confluence for 24 h prior to plating to allow completion of repair DNA synthesis. The GM2996 (XPC) cells show 0% UDS activity either before or after UV treatment (26) .

 
Progression from G1 into S-phase was then monitored in UV-irradiated cells that were allowed to repair their DNA prior to growth stimulation. As shown in Fig. 4B,Citation GM6419 cells that were allowed to complete DNA repair prior to growth stimulation (held in G1 for 24 h after UV treatment) were resistant to a UV-induced G1 arrest. Furthermore, UV radiation caused a complete G1-phase arrest in XPC cells, regardless of whether the cells were held in G1 for 24 h prior to growth stimulation (Fig. 4B)Citation . These results are consistent with unrepaired DNA damage being the signal for a p53-dependent G1 arrest in UV-irradiated cells.

Stabilization of p53 during a UV-induced G1-Phase Block.
The increase in p53 levels after UV treatment results, in large part, from stabilization of the p53 protein (2 , 3) . If p53 is stabilized to halt proliferation and allow DNA repair, then p53 stability is expected to decrease when DNA repair is complete. To test this possibility, p53 stability was determined in cells that were either growth stimulated immediately after UV exposure or were allowed to complete DNA repair prior to growth stimulation. The half-life of p53 was ~30 min in nonirradiated cells 12 h after release from G1 (Fig. 5Citation , 0J). In contrast, p53 was stabilized (half-life extended to >2 h) in cells exposed to a UV dose of 8 J/m2 and stimulated immediately after UV treatment. Under these conditions, the cells underwent a complete G1-phase cell cycle arrest (Figs. 1Citation and 4)Citation . Importantly, the half-life of p53 was decreased to that of nonirradiated cells in cells that were UV irradiated but held in G1 for 24 h prior to plating. Under these conditions, UV-induced DNA damage was completely repaired, and the cells progressed with normal kinetics from G1 into S-phase (Fig. 4)Citation . These results establish an excellent correlation between p53 stability and a G1 phase arrest in UV-irradiated cells.



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Fig. 5. GM6419 cells in the G1-phase were either untreated (0J) or UV irradiated at a dose of 8 J/m2. The cells were either plated immediately after UV treatment or were held at confluence for 24 h prior to plating. Twelve h after plating, the cells were treated with 25 µg/ml cyclohexamide (CHX) to inhibit de novo protein synthesis. Left panel, p53 steady-state levels were monitored by immunoblot analysis at various time points after the addition of CHX. The rate at which p53 steady-state levels decline in CHX-treated cells is a measure of the protein half-life. Right panel, the immunoblots were quantitated on a phosphorimager. The level of p53 protein at the zero time point in each case was considered 100%, and the decrease in p53 protein levels after CHX treatment is plotted.

 
Finally, p53, p21, and MDM2 protein levels were determined in UV-irradiated cells that were either growth stimulated immediately after UV exposure or were held in G1 for 24 h prior to growth stimulation (Fig. 6)Citation . Levels of all three proteins were increased in UV-irradiated cells that were plated immediately after UV exposure and were arrested in G1. In these experiments, p53 was induced at 5 h after release from G1 in the UV-irradiated cells, whereas MDM2 and p21 protein levels were not increased until 10 h after release from G1. The levels of all three proteins decreased in UV-irradiated cells that were held for 24 h in G1 prior to plating and were resistant to the UV-induced G1 arrest. It should be noted that p53 levels were not decreased in UV-irradiated cells held in G1 for up to 34 h after treatment (Fig. 6)Citation , despite the fact that DNA repair was complete within 24 h of holding in G1 (Fig. 4)Citation . This suggests that in addition to the completion of DNA repair, destabilization of p53 also requires the release of cells from the G1 phase. The expression patterns for p53 and p21 in this experiment are consistent with the UV-induced G1 arrest resulting from activation of the p53-p21 growth arrest pathway. It was perhaps interesting that MDM2 displayed an expression pattern similar to that of p53 and p21. MDM2 can bind p53 and promote its rapid degradation, and current models suggest that the stabilization of p53 in DNA-damaged cells results from an inhibition of p53:MDM2 binding (27, 28, 29) . In Fig. 6B,Citation we examined the level of p53:MDM2 binding complexes in this experiment by coimmunoprecipitation. A large amount of MDM2 immunoprecipitated with p53 from cells, which were plated immediately after UV exposure and in which p53 was stabilized. p53:MDM2 complexes were not observed until 10 h after release of the UV-irradiated cells from G1, consistent with the increased MDM2 levels observed at this time point. The fact that p53 was stabilized with no obvious decrease in p53:MDM2 binding suggests that UV radiation may stabilize p53 through alternative pathways, in addition to inhibiting the interaction between p53 and MDM2.



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Fig. 6. A, GM6419 cells in the G1-phase were untreated (0J) or UV irradiated at a dose of 8 J/m2. The cells were either plated immediately after UV treatment or were held at confluence for 24 h prior to plating. At the indicated time points after plating, protein extracts were prepared and examined by immunoblot analysis for p53, p21, and MDM2. B, p53 was immunoprecipitated using the p53 antibody Ab-421 and examined by immunoblot analysis with the MDM2 antibody SMP-14 to detect p53:MDM2 binding complexes. *, position of the antibody heavy chain used in the immunoprecipitation.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
When normal mammalian cells are exposed to DNA-damaging agents, they undergo a transient G1- and G2-phase cell cycle arrest. These arrests allow cells time to repair the damaged DNA before proceeding with either replicative DNA synthesis or mitosis. Failure to arrest in either G1 or G2 phase could lead to an accumulation of mutations because of the replication of a damaged genome. IR induces a G1 arrest in cells expressing wild-type p53 but not in cells that either lack p53 expression or in which p53 is inactivated (4, 5, 6) . These results demonstrate an essential role for p53 in the cell cycle response to IR. In contrast to IR, however, a clear role for p53 in the cell cycle response to certain other DNA-damaging agents has not been established. For example, UV radiation inhibited cell cycle progression in normal embryonic stem (ES) cells and in ES cells homozygous for a targeted deletion of p53 (30) . Furthermore, high doses of either UV radiation or actinomycin D were reported to induce a G1 arrest in cells with wild-type p53 and in cells in which p53 was inactivated by expression of the HPV-16 E6 oncoprotein (12) . Finally, a p53-independent G1 arrest was reported in murine 3T3 cells exposed to the DNA modifying agent benzo(a)pyrene (31) . These finding indicate that certain DNA-damaging agents can signal a G1 cell cycle arrest through mechanisms that are independent of p53.

The purpose of this study was to examine the role and regulation of p53 during a UV-induced G1 arrest. A dose-dependent G1 arrest was observed in normal human fibroblasts as well as in fibroblasts deficient in the repair of UV-induced DNA damage. Expression of HPV16-E6, which promotes the degradation of p53, or a dominant-negative p53 mutant that inactivates wild-type p53, caused the cells to become resistant to this UV-induced arrest. These results clearly demonstrate that p53 can activate a G1 cell cycle arrest in response to UV radiation. Interestingly, however, cells in which p53 was inactivated still underwent a significant G1 to S-phase delay after UV treatment. These findings indicate that UV radiation can also activate a G1 delay that is independent of wild-type p53. On the basis of these findings, we suggest that UV radiation affects multiple pathways to cause a G1-phase arrest or delay, only one of which involves p53.

Our results suggest that the p53-dependent G1 arrest in UV-irradiated cells results from UV damage to actively transcribed genes. This is based on the fact that the minimum UV dose that caused a complete G1 arrest in uninfected or control virus-infected cells was 8 J/m2 in cells with normal DNA repair capacity (GM6419 cells), 5–6 J/m2 in XPC cells, and 1.5–2.0 J/m2 in XPD cells. Thus, XPD cells, which are deficient in the repair of actively transcribed genes, are more susceptible to a UV-induced G1 arrest than are either XPC cells or normal cells, which are not compromised in the repair of transcribed DNA strands. In this regard, it is worth noting the studies of Ford and Hanawalt (15) in which wild-type p53 was required for efficient repair of nontranscribed DNA regions but not for repair of transcribed DNA strands. Insofar as p53 is not required for repair of actively transcribed genes, these results would suggest that the induction of p53 through UV damage to actively transcribed genes is independent of its role in DNA repair. Other studies support our notion that UV radiation signals to p53 through damage to actively transcribed genes. For example, the MRD that stabilized p53 was estimated in normal cells and in cells deficient in various aspects of DNA repair (32) . The MRD in cells specifically deficient in the repair of actively transcribed genes was 8-fold lower than the MRD of cells with normal DNA repair capacity. In contrast, the MRD for cells specifically deficient in the repair of nontranscribed DNA regions was as high as that of normal cells. These results suggested that DNA damage to actively transcribed genes is the signal for the stabilization of p53 in response to UV radiation.

The mechanism by which UV induces a p53-independent G1 to S-phase delay is unknown. A recent study suggested that high doses of UV radiation can inhibit the expression of E2F-1-transactivated gene products that are required for G1 to S-phase progression (12) . Thus, decreased expression of these E2F-1-regulated genes could contribute to the p53-independent G1 to S-phase delay observed in the current report. In a separate study, UV radiation was reported to induce the expression of p21 and a concomitant G1 arrest in Li-Fraumeni cells that lacked both p53 alleles (33) . Although this induction of p21 may explain the p53-independent responses to UV radiation in some systems, we did not observe an induction of p21 in UV-irradiated cells in which p53 was inactivated. It is worth noting that in our study, XPD cells in which p53 was inactivated by a p53 dominant-negative mutant remained more sensitive to a UV-induced G1 arrest than either XPC or normal cells in which p53 was similarly inactivated. These results suggest that damage to actively transcribed genes may be the signal for a p53-independent G1-phase delay, in addition to the p53-dependent arrest.

The mechanism by which UV radiation and other DNA-damaging agents stabilize p53 has not been fully clarified. MDM2 can bind p53 and promote its rapid degradation through the ubiquitin proteolysis pathway (27 , 28) . Current models suggest that DNA-damaging agents stabilize p53 by inhibiting p53:MDM2 binding (29) . According to this model, one would predict a decreased interaction between p53 and MDM2 under DNA-damaging conditions that stabilize p53. In the current study, MDM2 protein levels were increased under conditions that stabilized p53, and the UV-irradiated cells underwent a G1 cell cycle arrest. Interestingly, the increase in MDM2 levels coincided with a corresponding increased level of p53:MDM2 binding complexes. These results raise the possibility that UV may affect multiple pathways to stabilize p53, in addition to inhibiting the interaction between p53 and MDM2.

Ineffective repair of UV-induced DNA damage can result in a high predisposition to cancer, as well as an increased sensitivity to UV-induced cell death (34 , 35) . Thus, efficient DNA repair after exposure to UV radiation is essential for maintaining normal cellular homeostasis. The current study indicates that UV can induce a G1 cell cycle arrest or delay through p53-dependent and -independent mechanisms. Furthermore, our results suggest that unrepaired DNA damage to actively transcribed genes is the likely signal for a p53-dependent G1 arrest. The presence of multiple pathways for activating a G1 arrest or delay in response to UV radiation underlies the potential importance of such an arrest in the DNA repair response.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Strains and Retroviral Infections.
All cell types used in this study were maintained in DMEM containing 15% fetal bovine serum. The human diploid fibroblast strains GM6419, the XPC cell strains GM2995 and GM2996, and the XPD cell strains GM03247 and GM0524 were obtained from the Corrielle cell repository in Camden, NJ. GM6419 cells have normal repair capacity for UV-induced DNA lesions. Cell lines producing the HPV-16 E6 or control retrovirus (LXSN) were obtained from Denise Galloway (University of Washington, Seattle, WA). The DNA construct for production of the dominant-negative p53 retrovirus (referred to as p53-CTF) was obtained from Moshe Oren (Weizmann Institute of Science, Rehovot, Israel). p53-CTF encodes the COOH-terminal oligomerization domain of p53 and inactivates wild-type p53 in infected cells (10) . The p53-CTF retrovirus-producing cell line was generated by Alan Thompson (Harvard Medical School). Retroviral infection was carried out by incubating exponentially growing GM6419, XPC, or XPD cells in 4 ml of medium containing a 1-ml aliquot of each retrovirus and 4 µg/ml Polybrene for 4 h. The cells were then rinsed with fresh medium once and refed with fresh medium and incubated overnight. The cells were then split at a dilution of approximately 1:4 and maintained in normal medium for an additional 24 h, at which point the cells were refed with medium containing 200 µg/ml G418. The cells were maintained in G418-containing medium for 2 weeks, and pooled populations of selected cells were obtained.

UV Radiation Treatment and Cell Cycle Analysis.
UV irradiation was carried out as described previously (2) . The UV light exposure apparatus consisted of five UV bulbs in a specially constructed incubator that delivered 254 nm light at a dose of 2.08 J/m2/s. Confluent, G1-phase cells were rinsed with PBS and exposed to the indicated UV dose. The cells were then trypsinized and replated at low density to stimulate their movement from G1 into S-phase. At the indicated time after growth stimulation, cells were trypsinized and fixed in 70% ethanol. The fixed cells were suspended in PBS containing 1 mg/ml propidium iodide and 1000 Kunitz units/ml RNase A. Cell cycle distribution was determined by FACS analysis at the Dana-Farber Flow Cytometry Laboratory.

Western Blots, Immunoprecipitations, and p53 Stability Measurements.
For Western blot analysis, cells were washed twice with PBS, scraped into 0.5 ml of lysis buffer [50 mM Tris (pH 8.0), 5 mM EDTA, 150 mM NaCl, 0.5% NP40, and 1 mM phenylmethylsulfonyl fluoride], and incubated on ice for 15 min with occasional light vortexing. Lysates were spun at 15,000 x g for 15 min to remove cellular debris. Protein extract from the resulting supernatant was resolved by SDS-PAGE and transferred to Immobilon-P membranes (Millipore) for detection with either the Ab-6 p53 antibody (Oncogene Science), the anti-p21 polyclonal antibody 15431E (PharMingen), or the anti-MDM2 antibody SMP-14. For analysis of p53:MDM2 binding, p53 was immunoprecipitated from lysates using the p53 antibody Ab-421 (Oncogene Science) and subsequently examined by immunoblot analysis using the MDM2 antibody SMP-14.

DNA Repair Measurements.
DNA repair activity (UDS) was assessed as described (26) in UV-irradiated GM6419 cells in the following manner. G1 phase cells were UV irradiated (8 J/m2) and maintained in G1 for 24 h. At various time points after UV treatment, the cells were pulse labeled with 10 µCi [3 H]thymidine. Because the cells were in G1, the uptake of [3 H]thymidine in the general cell population was attributable to DNA repair synthesis only and not attributable to replicative DNA synthesis. The uptake of radionucleotide at each time point was monitored by fixing the cells directly to the culture dish and subsequently exposing them to a photographic emulsion prior to autoradiographic development. The average number of silver grains precipitated from the emulsion per cell nucleus was determined by microscopic examination and was used as a measurement of DNA repair activity. The experiment was performed in duplicate, and the average number of silver grains precipitated per cell nucleus at each time point was determined. The highest number of silver grains were precipitated from each cell nucleus immediately after UV treatment.


    Acknowledgments
 
We acknowledge Peter M. Howley for guidance and critical reading of the manuscript.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by USPHS Grant 1R01CA80918 and by a breast cancer research grant from the Massachusetts Department of Public Health (both to C. G. M.). Back

2 To whom requests for reprints should be addressed, at Harvard School of Public Health, Department of Cancer Cell Biology, 665 Huntingdon Avenue, Building 1, Second Floor, Boston, MA 02115. Phone: (617) 432-2532; Fax: (617) 432-2640; E-mail: cmaki{at}hsph.harvard.edu Back

3 The abbreviations used are: IR, ionizing radiation; NER, nucleotide excision repair; XP, xeroderma pigmentosa; HPV, human papillomavirus; UDS, unscheduled DNA synthesis; MRD, minimum required UV dose; FCS, fluorescence-activated cell sorting. Back

Received for publication 9/22/99. Revision received 1/24/00. Accepted for publication 1/24/00.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

  1. Levine A. J. p53, the cellular gatekeeper for growth and division. Cell, 88: 323-331, 1997.[Medline]
  2. Maki C. G., Howley P. M. Ubiquitination of p53 and p21 is differentially affected by ionizing and UV radiation. Mol. Cell. Biol., 17: 355-363, 1997.[Abstract/Free Full Text]
  3. Maltzman W., Czyzyk L. UV irradiation stimulates levels of p53 cellular tumor antigen in nontransformed mouse cells. Mol. Cell. Biol., 4: 1689-1694, 1984.[Abstract/Free Full Text]
  4. Tsang N-M., Nagasawa H., Li C., Little J. B. Abrogation of p53 function by transfection of HPV16 E6 gene enhances the resistance of human diploid fibroblasts to ionizing radiation. Oncogene, 10: 2403-2408, 1995.[Medline]
  5. Kuerbitz S. J., Plunkett B. S., Walsh W. V., Kastan M. B. Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc. Natl. Acad. Sci. USA, 89: 7491-7495, 1992.[Abstract/Free Full Text]
  6. Kessism T. D., Slebos R. J., Nelson W. G., Kastan M. B., Plunkett B. S., Han S. M., Lorincz A. T., Hedrick L., Cho K. R. Human papillomavirus 16 E6 expression disrupts the p53-mediated cellular response to DNA damage. Proc. Natl. Acad. Sci. USA, 90: 3988-3992, 1993.[Abstract/Free Full Text]
  7. Havre P. A., Yuan J., Hedrick L., Cho K. R., Glazer P. M. p53 inactivation by HPV16 E6 results in increased mutagenesis in human cells. Cancer Res., 55: 4420-4424, 1995.[Abstract/Free Full Text]
  8. Yu Y., Li C. Y., Little J. B. Abrogation of p53 function by HPV16 E6 gene delays apoptosis and enhances mutagenesis but does not alter radiosensitivity in TK6 human lymphoblast cells. Oncogene, 14: 1661-1667, 1997.[Medline]
  9. Lowe S. W., Schmitt E. M., Smith S. W., Osborne B. A., Jacks T. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature (Lond.), 362: 847-849, 1993.[Medline]
  10. Eizenberg O., Faber-Elman A., Gottlieb E., Oren M., Rotter V., Schwartz M. p53 plays a regulatory role in differentiation and apoptosis of central nervous system-associated cells. Mol. Cell. Biol., 16: 5178-5185, 1996.[Abstract/Free Full Text]
  11. Haapajarvi T., Kivinen L., Pitkanen K., Laiho M. Cell cycle dependent effects of UV-radiation on p53 expression and retinoblastoma protein phosphorylation. Oncogene, 11: 151-159, 1995.[Medline]
  12. Chang D., Chen F., Zhang F., McKay B. C., Ljungman M. Dose-dependent effects of DNA-damaging agents on p53-mediated cell cycle arrest. Cell Growth Differ., 10: 155-162, 1999.[Abstract/Free Full Text]
  13. Gujuluva C. N., Baek J. H., Shin K. H., Cherrick H. M., Park N. H. Effect of UV-irradiation on cell cycle, viability, and the expression of p53, gadd153 and gadd45 genes in normal and HPV-immortalized human oral keratinocytes. Oncogene, 9: 1819-1827, 1994.[Medline]
  14. Friedberg E. C., Walker G. C., Siede W. DNA repair and mutagenesis283-366, ASM Press Washington, DC 1995.
  15. Ford J. M., Hanawalt P. C. Expression of wild-type p53 is required for efficient global genomic nucleotide excision repair in UV-irradiated human fibroblasts. J. Biol. Chem., 272: 28073-28080, 1997.[Abstract/Free Full Text]
  16. Schaeffer L., Moncollin V., Roy R., Staub A., Mezzina M., Sarasin A., Weeda G., Hoeijmakers J. H. J., Egly J-M. The ERCC2/DNA repair protein is associated with the class II BTF2/TFIIh transcription factor. EMBO J., 13: 2388-2392, 1994.[Medline]
  17. Schaeffer L., Roy R., Humbert S., Moncollin V., Vermuellen W., Hoeijmakers J. H. J., Chambon P., Egly J-M. DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor. Science (Washington DC), 260: 58-63, 1993.[Abstract/Free Full Text]
  18. Wang X. W., Yeh H., Schaeffer L., Roy R., Moncollin V., Egly J-M., Wang Z., Freidberg E. C., Evans M. K., Taffe B. G., Bohr V. A., Weeda G., Hoeijmakers J. H. J., Forrester K., Harris C. C. p53 modulation of TFIIH-associated nucleotide excision repair activity. Nat. Genet., 10: 188-195, 1995.[Medline]
  19. Wang X. W., Vermeullen W., Coursen J. D., Gibson M., Lupold S. E., Forrester K., Xu G., Elmore L., Yeh H., Hoeijmakers J. H., Harris C. C. The XPB and XPD DNA helicases are components of the p53-mediated apoptosis pathway. Genes Dev., 10: 1219-1232, 1996.[Abstract/Free Full Text]
  20. Scheffner M., Werness B. A., Huibregtse J. M., Levine A. J., Howley P. M. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell, 63: 1129-1136, 1990.[Medline]
  21. Scheffner M., Huibregtse J. M., Vierstra R., Howley P. M. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell, 75: 495-505, 1993.[Medline]
  22. Scheffner M., Nuber U., Huibregtse J. M. Protein ubiquitination involving an E1–E2-E3 enzyme ubiquitin thioester cascade. Nature (Lond.), 373: 81-83, 1995.[Medline]
  23. Vermeulen W., de Boer J., Citterio E., van Gool A. J., van der Horst G. T., Jaspers N. G., de Laat W. L., Sijbers A. M., van der Spek P. J., Sugasawa K., Weeda G., Winkler G. S., Bootsma D., Egly J-M., Hoejmakers J. H. Mammalian nucleotide excision repair and syndromes. Biochem. Soc. Trans., 25: 309-315, 1997.[Free Full Text]
  24. Venema J., van Hoffen A., Natarajan A. T., van Zeeland A. A., Mullenders L. H. F. The residual repair capacity of xeroderma pigmentosum complementation group C fibroblasts is highly specific for transcriptionally active DNA. Nucleic Acids Res., 18: 443-448, 1990.[Abstract/Free Full Text]
  25. Cullinane C., Weber C. A., Dianov G., Bohr V. A. Restoration of preferential and strand specific gene repair in group 2 Chinese hamster ovary mutants (UV5) by the XPD (ERCC2) gene. Carcinogenesis (Lond.), 18: 681-686, 1997.[Abstract/Free Full Text]
  26. Nagasawa H., Burke M. J., Little F. F., McCone E. F., Chan G. L., Little J. B. Multiple abnormalities in the ultraviolet light response of cultured fibroblasts derived from patients with the basal cell nevus syndrome. Teratog. Carcinog. Mutagen., 8: 25-33, 1988.[Medline]
  27. Haupt Y., Maya R., Kazaz A., Oren M. Mdm2 promotes the rapid degradation of p53. Nature (Lond.), 387: 296-299, 1997.[Medline]
  28. Kubbutat M. H., Jones S. N., Vousden K. H. Regulation of p53 stability by MDM2. Nature (Lond.), 387: 299-303, 1997.[Medline]
  29. Shieh S-Y., Ikeda M., Taya Y., Prives C. DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell, 91: 325-334, 1997.[Medline]
  30. Prost S., Bellamy C. O., Clarke A. R., Wyllie A. H., Harrison D. J. p53-independent DNA repair and cell cycle arrest in embryonic stem cells. FEBS Lett., 425: 499-504, 1998.[Medline]
  31. Vaziri C., Faller D. V. A benzo[a]pyrene-induced cell cycle checkpoint resulting in p53-independent G1 arrest in 3T3 fibroblasts. J. Biol. Chem., 272: 2762-2769, 1997.[Abstract/Free Full Text]
  32. Yamaizumi M., Sugano T. UV-induced nuclear accumulation of p53 is evoked through DNA damage of actively transcribed genes independent of the cell cycle. Oncogene, 9: 2775-2784, 1994.[Medline]
  33. Loignon M., Fetni R., Gordon A. J., Drobetsky E. A. A p53-independent pathway for induction of p21waf1cip1 and concomitant G1 arrest in UV-irradiated human skin fibroblasts. Cancer Res., 57: 3390-3394, 1997.[Abstract/Free Full Text]
  34. Lommel L., Hanawalt P. C. Increased UV resistance of a xeroderma pigmentosum revertant cell line is correlated with selective repair of the transcribed strand of an expressed gene. Mol. Cell. Biol., 13: 970-976, 1993.[Abstract/Free Full Text]
  35. Cheo D. L., Meira L. B., Hammer R. E., Burns D. K., Doughty A. T., Friedberg E. C. Synergistic interactions between XPC and p53 mutations in double-mutant mice: neural tube abnormalities and accelerated UV radiation-induced skin cancer. Curr. Biol., 6: 1691-1694, 1996.[Medline]



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