UV-induced DNA incision and proliferating cell nuclear antigen recruitment to repair sites occur independently of p53replication protein A interaction in p53 wild type and mutant ovarian carcinoma cells
Federica Riva,
Valentina Zuco1,
Ard A. Vink2,
Rosanna Supino1 and
Ennio Prosperi,3
Centro di Studio per l'Istochimica del CNR, Piazza Botta 10, 27100 Pavia,
1 Divisione di Oncologia Sperimentale B, Istituto Tumori, Milano, Italy and
2 TNO Nutrition and Food Research Institute, Zeist, The Netherlands
 |
Abstract
|
---|
The tumour suppressor gene TP53 plays an important role in the regulation of DNA repair, and particularly of nucleotide excision repair. The influence of p53 status on the efficiency of the principal steps of this repair pathway was investigated after UV-C irradiation in the human ovarian carcinoma cell line IGROV-1 (expressing wild-type p53) and in the derived clone IGROV-1/Pt1 (with p53 mutations at codons 270 and 282). Clonogenic survival after UV-C irradiation showed that IGROV-1/Pt1 cells were ~2-fold more resistant to DNA damage than parental cells. Modulation of p53 protein levels, cell cycle arrest and apoptosis were induced in UV-irradiated IGROV-1 cells, but not in the p53-mutant cell line. Exposure to UV or cisplatin induced down-regulation of p53replication protein A (RPA) interaction in parental, but not in IGROV-1/Pt1 cells. However, persistent binding of p53 to RPA did not affect the early steps of DNA repair. In fact, both UV-induced DNA incision and the recruitment of proliferating cell nuclear antigen (PCNA) to DNA repair sites occurred to a comparable extent in p53-wild type and -mutant cell lines, although PCNA remained associated with chromatin for a longer period of time in IGROV-1/Pt1 cells. Global genome repair, as detected by immunoblot analysis of cyclobutane pyrimidine dimers, was not significantly different in the two cell lines at 3 h after UV irradiation. In contrast, lesion removal at 24 h was markedly reduced in IGROV-1/Pt1 cells, being ~25% of the initial amount of damage, as compared with ~50% repair in parental cells. These results indicate that the presence of mutant p53 protein and its persistent interaction with RPA do not affect the early steps of nucleotide excision repair in IGROV-1/Pt1 cells. Thus, repair defects in p53-mutant ovarian carcinoma cells may be attributed to late events, possibly related to a reduced removal/recycling of PCNA at repair sites.
Abbreviations: AraC, 1-ß-D-arabinofuranosylcytosine; BER, base excision repair; CPD, cyclobutane pyrimidine dimer; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; HU, hydroxyurea; LFS, Li; raumeni syndrome; NER, nucleotide excision repair; PCNA, proliferating cell nuclear antigen; PI, propidium iodide; RPA, replication protein A; XP, xeroderma pigmentosum.
 |
Introduction
|
---|
DNA repair efficiency is an important determinant of the cell response to DNA damaging agents (1,2), since the inability to eliminate the damage may affect genomic stability, thereby leading to mutagenesis (3,4). Inactivation of the p53 tumour suppressor gene was shown to affect the cellular response to DNA damage (5,6). In fact, disruption of p53 function by mutation or expression of the viral E6 protein was found to reduce repair of UV-induced DNA damage (7). Defective DNA repair was documented in SV40-transformed human fibroblasts and in LiFraumeni syndrome (LFS) cells with a mutant p53 gene (810). More recent results have shown that cells with a homozygous mutation in the p53 gene and fibroblasts expressing the HPV E6 gene were deficient in global nucleotide excision repair (NER), but not in transcription-coupled repair, i.e. the repair of the transcribed strand in active genes (1113). Other studies reported an impairment of both NER sub-pathways in various cell lines (1418). Thus, it was suggested that p53 could play a role in DNA repair (19) and a direct involvement in the base excision repair (BER) process has recently been demonstrated (20,21). In the case of NER, a function for p53 has been proposed on the basis of its interaction with RPA (22) and with the proteins XPB and XPD (14), whose activities, however, are inhibited by p53. It has been suggested that the interaction with RPA sequesters p53 and this interaction is disrupted after UV-induced DNA damage (23). Interestingly, this interaction is not disrupted upon DNA damage in cells deficient in global NER (23), thus suggesting that release of p53 and RPA may be necessary for NER. However, it is not yet clear whether p53 plays a direct role in NER, or its influence occurs predominantly at the transcriptional level, by affecting the expression of factors participating in the repair process (19).
The NER pathway is characterized by three major steps: (i) recognition of the damage; (ii) DNA incision and excision; and (iii) DNA re-synthesis and ligation (24,25). A general model of NER suggests that the XPC and hHR23B proteins together act as a damage sensor complex (26). Then the transcription factor TFIIH, containing the XPB and XPD helicases, acts to separate the DNA strands, while XPA binds the open DNA and replication protein A (RPA) stabilizes this conformation. Alternatively, XPA and RPA are proposed to act themselves as sensors of damage (27). Subsequently, ERCC1-XPF and XPG endonucleases excise a ~30 nucleotide fragment that will be reconstructed by DNA polymerases
/
, in a reaction dependent on the proliferating cell nuclear antigen (PCNA) and the repair intermediates will be finally closed by DNA ligase I (28).
In this study, we have investigated whether the efficiency of these steps may be influenced by the interaction of a mutant p53 with RPA protein. In particular, DNA incision and the subsequent recruitment of DNA synthesis proteins were considered. In fact, RPA is required for efficient DNA incision (29,30). Similarly, the recruitment of PCNA to DNA repair sites was analysed because this step was previously shown not to occur, or to be delayed, in xeroderma pigmentosum (XP) cells of group A and G, which are deficient in DNA incision (3133). In addition, PCNA plays a central role in NER not only by participating in DNA synthesis (34,35), but also by coordinating DNA repair with cell cycle control (36,37).
Recruitment of PCNA to DNA repair sites involves the transition of the protein from a soluble to a chromatin-bound and detergent-insoluble state (38). This feature is a useful marker of the ongoing DNA re-synthesis and consequently it determines also the ability of the cells to perform the repair steps preceding PCNA activation (34,35).
The human ovarian carcinoma cell line IGROV-1 was used as the model system together with the p53-mutant derivative IGROV-1/Pt1, obtained by cisplatinum selection of a spontaneously mutated population of IGROV-1 (39). IGROV-1/Pt1 cells were previously shown to contain a p53 gene mutated at codons 270 and 282, thereby expressing a transcriptionally inactive protein (40). The results show that in IGROV-1/Pt1 cells the interaction between p53 and RPA is not lost after UV or cisplatin-induced DNA damage, as is the case in parental cells. However, p53 sequestration by RPA did not affect the early steps of NER, since p53-mutant cells showed an incision activity and PCNA recruitment comparable with that of parental IGROV-1 cells. In contrast, defective removal of cyclobutane pyrimidine dimers (CPDs) was observed in IGROV-1/Pt1 cells at late times after DNA damage and found to be temporally coincident with high levels of PCNA remaining associated with chromatin.
 |
Materials and methods
|
---|
Cell cultures and treatments
The ovarian carcinoma cell line IGROV-1 and the derivative IGROV-1/Pt1 were grown in RPMI 1640 medium (BioWhittaker, Verviers, Belgium) supplemented with 10% fetal bovine serum (Gibco-BRL, Paisley, UK). A detailed characterization of the p53-mutant cell clone has been reported elsewhere (39,40). Cells were plated in 100-mm Petri dishes and grown for 48 h before treatments. Cells were irradiated in sterile conditions with a T-UV9 UV-C germicidal lamp (Philips, Eindoven, Netherlands) at a fluence rate of ~0.5 J/m2/s at 254 nm. Delivered dose was measured with a radiometer (Spectronics, Westbury, NY). Cells were washed in sterile phosphate-buffered saline (PBS) before irradiation and placed in complete medium immediately after exposure for the required periods of time.
Clonogenic survival, apoptosis and cell cycle analysis
Cells were seeded at a density of 1x103 in 100-mm plates in triplicate and UV irradiated 24 h after seeding. After culture for 5 days in growth medium, cells were rinsed with PBS, fixed with methanol and stained with crystal violet. Colonies with at least 50 cells were scored with an inverse microscope and surviving fractions were calculated.
For determination of apoptosis, attached and floating cells were collected at various time points after irradiation, fixed in 70% ethanol and stained with 30 µg/ml propidium iodide (PI) in PBS containing 1 mg/ml RNAse A (Sigma, Milan, Italy). Cells with morphological features of apoptosis, like chromatin condensation and margination (11), were counted with a Leitz Orthoplan fluorescence microscope for determination of apoptotic index.
For cell cycle analysis, 104 cells stained with PI, as described above, were measured with an Epics XL flow cytometer equipped with an argon laser for fluorescence excitation at 488 nm (Coulter, Miami, FL). Statistical analysis of the percentage of cells in each phase of the cell cycle was performed by means of the XL2 software (Coulter).
Western blot analysis of p53 protein and co-immunoprecipitation of RPA protein
Cells grown as described above were irradiated with UV-C light, or treated for 1 h with cisplatin, and then harvested at different periods of times after treatment. Cells were dissolved directly in SDS sample buffer (65 mM TrisHCl pH 8.0, 65 mM ß-mercaptoethanol, 1% SDS and 10% glycerol) and 100 µg protein was loaded on a 10% SDSpolyacrylamide gel and electrophoresed. Proteins were then transferred onto a nitrocellulose filter (Amersham, Little Chalfont, UK). The membrane was incubated in PBSTween (PBS containing 0.2% Tween 20 and 5% non-fat dried milk to block unspecific binding of antibody) and incubated for 1 h at room temperature with monoclonal antibody to p53 (DO-7; Dako, Copenhagen, Denmark), or with anti-actin antibody (Sigma). The membrane was then washed with PBSTween solution and incubated for 30 min with anti-mouse horseradish (HRP)-conjugated antibody (Sigma) diluted 1:5000. At the end of incubation, the membrane was washed as above and detection of the bands was performed with chemiluminescence using the ECL detection kit (Amersham).
For p53 immunoprecipitation, ~5x106 cells/sample were lysed in extraction buffer (50 mM TrisHCl pH 7.4, 0.5% Nonidet P-40, 1 mM EDTA, 0.2 mM sodium vanadate and protease inhibitors). Samples were centrifuged at 13 000 g to clear cell lysates and then cell extracts were incubated for 1 h at room temperature with 1 µg p53 antibody (Ab-3; Oncogene Science, Cambridge, MA) and then overnight at 4°C. Protein ASepharose (100 µl; Pharmacia, Uppsala, Sweden) were then added to each sample, and incubated for 1 h at room temperature. Samples were then centrifuged at 13 000 g and washed four times with extraction buffer. The final pellet was resuspended in 60 µl SDS sample buffer and boiled for 5 min. After separation in 12% SDSPAGE, proteins were blotted onto polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA) and finally probed with 9H8 monoclonal antibody against the RPA 32-kDa subunit (kindly provided by Prof. M.Wold, Iowa University). The membrane was developed with ECL, as described above.
Determination of DNA incision by alkaline elution
Cells (1x105/cm2) were labelled for 30 h with 0.08 µCi/ml [14C]thymidine (56 mCi/mmol specific activity; Amersham). Cell cultures were post-incubated for 18 h in the absence of labelled thymidine to chase the DNA-incorporated radioactivity. Cells were pre-incubated for 30 min in medium containing 100 µM 1-ß-D-arabinofuranosylcytosine (araC; Sigma) and 10 mM hydroxyurea (HU; Sigma) before irradiation with 10 J/m2, to prevent rejoining after DNA incision (41). A positive control of cells irradiated with
-rays (8 Gy) was also used as reference. At 5 min, or 3 h after UV exposure, untreated control and irradiated samples were harvested by mild trypsinization and processed for alkaline elution according to the method of Fornace (42). Briefly, 7x105 cells were loaded onto a 2 µm pore size Nucleopore polycarbonate filters (Costar, Milan, Italy) and lysed with an SDSEDTA lysing solution (2% SDS, 25 mM Na2EDTA, 100 mM glycine, pH 10) to which 0.5 mg/ml proteinase K (Merck, Darmstadt, Germany) were added. After lysis, the eluting solution (20 mM H4EDTA and 10% ammonium tetrapropylhydroxide in water, pH 12.15) was added. Fractions were collected at 180 min intervals with an automatic fraction collector linked to a peristaltic pump running at 0.035 ml/min. Radioactivity of collected fractions was counted with a 1414 Winspectral (Wallac EG&G, Turku, Finland) liquid scintillation ß-counter and elution profiles were determined. Elution rate constants were calculated as described (43).
Western blot and immunocytometric determination of PCNA recruitment
Irradiated and control cells (~3x106/sample) were harvested, washed in PBS and lysed in cold hypotonic solution to release soluble PCNA (44). Briefly, cells were lysed in ice-cold 10 mM TrisHCl buffer (pH 7.4) containing 2.5 mM MgCl2, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonylfluoride (PMSF), 0.2 mM Na3VO4 and a cocktail of protease inhibitors (Sigma).
For western blot analysis, lysed cells were centrifuged to collect the soluble fraction and then incubated for 30 min at 37°C with 300 U DNAse I (Sigma) in 10 mM TrisHCl (pH 7.4) containing 5 mM MgCl2 and 10 mM NaCl to release the DNA-bound form of PCNA (45). Each soluble and DNAse-released fraction was mixed with SDS loading buffer, and analysed by SDSPAGE and blotting, as described above. PCNA was detected with PC10 antibody (Dako), followed by a secondary HRP-conjugated antibody, as previously described (45).
For immunofluorescence determination, ~1x106 cells were centrifuged after hypotonic lysis, resuspended in cold physiological saline and fixed by adding cold (20°C) ethanol to a final concentration of 70%. Cells were washed in PBS, incubated for 15 min at room temperature in blocking solution (PBS containing 1% bovine serum albumin and 0.2% Tween 20) to prevent unspecific staining. After that, cells were incubated for 1 h in 100 µl anti-PCNA monoclonal antibody diluted to 5 µg/ml. The samples were then washed three times (10 min each) in blocking solution and further incubated for 30 min in FITC-conjugated anti-mouse secondary antibody (Sigma) diluted 1:100. At the end of the reaction, cells were again washed and resuspended in PBS containing 5 µg/ml PI and 1 mg/ml RNAse A. Cells were analysed with a flow cytometer, as described above, and a total of 1x104 cells were measured for each sample. Fluorescence intensity signals of PCNA immunostaining in each phase of the cell cycle was evaluated by gating the cells according to their DNA content value, as determined by PI fluorescence. Electronic gating provided by the instrument software XL II (Coulter) was used for exclusion of cell doublets.
Immunoblot analysis of CPD removal
Cells (5x106/sample) were irradiated with UV light at 10 J/m2 and then grown for various periods of time (024 h). Cells were harvested, washed in PBS and frozen (80°C) until use. For DNA extraction, cells were lysed in 0.5 ml 10 mM TrisHCl (pH 8.2), 2 mM EDTA, 0.5% SDS and 1 mg/ml proteinase K (Sigma). Samples were incubated overnight at 37°C and DNA was extracted with phenolchloroform and ethanol precipitation. DNA recovered from each sample was quantified by absorbance at 260 nm prior to denaturation at 100°C for 5 min and chilling on ice. Denatured DNA (400 ng) was loaded onto nitrocellulose using a slot-blot apparatus (Schleicher & Schuell, Dassel, Germany). The membranes were dried for 2 h at 70°C, blocked for 30 min in PBSTween solution and then incubated for 1 h in H3 mouse monoclonal antibody (1:1000 dilution) against CPDs (46). Antibody binding was detected by a biotin-conjugated anti-mouse antibody (Sigma) diluted 1:2000, followed by HRP-conjugated streptavidin (Amersham) diluted 1:1000. Peroxidase reaction was developed with ECL, as described above. Samples of denatured calf thymus DNA (Sigma), UV irradiated with 0, 10, 25 and 50 J/m2, were included as a positive control in each immunoblot experiment. Quantitation of the immunoreactive signals was performed by densitometry, utilizing the NIH image analysis 1.60 software (12).
 |
Results
|
---|
UV-C response of wild-type and p53 mutant IGROV-1 cells
The sensitivity to UV-C irradiation of the two ovarian carcinoma cell lines was evaluated with a standard clonogenic assay. The results reported in Figure 1
show that IGROV-1/Pt1 cells were more resistant to UV-C than parental cells, with an estimated IC50 of 5 J/m2 being 2-fold higher than that of IGROV-1 cells (2.5 J/m2).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 1. Sensitivity of IGROV-1 ( ) and IGROV-1/Pt1 () cells to UV-C irradiation, as determined by clonogenic survival following UV-C irradiation at different doses (J/m2). The data are expressed as percentage of colonies formed in irradiated samples as compared with unirradiated controls. Mean values ± SEM of three independent experiments are shown
|
|
Western blot analysis of p53 protein levels confirmed the modulation of wild-type protein in UV-irradiated IGROV-1 cells, but not of the p53-mutant protein in IGROV-1/Pt1 cells (Figure 2A
). The lack of transcriptional activation of mutant p53 in IGROV-1/Pt1 cells after UV irradiation was also verified by assessing the induction of cell cycle changes and of apoptosis. Figure 2B
shows the effects induced by UV-C irradiation on cell cycle progression, as determined by flow cytometric analysis of DNA content. IGROV-1 cells showed a significant reduction (from about 25 to 12%) in the number of cells in S-phase at 24 (data not shown) and 48 h after UV irradiation. However, a typical G1 arrest could not be easily detected, due to the evident induction of cell death, as indicated by the presence of cells in the sub-G1 peak region of the DNA histogram. In contrast, UV-irradiated IGROV-1/Pt1 cells did not show any significant variation in cell cycle phase distribution with respect to untreated control samples. The morphological analysis by fluorescence microscopy confirmed that, as compared with untreated controls, a significant increase (~25%) in the number of cells showing typical features of apoptosis could be already observed 24 h after irradiation in parental IGROV-1 cells. In contrast, no significant induction of apoptotic cell death was found in IGROV-1/Pt1 cells (Figure 2C
).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2. Response of IGROV-1 and IGROV-1/Pt1 cells to UV-C irradiation. (A) Western blot analysis of p53 protein levels at 8 and 24 h after UV exposure. Samples were also blotted with an antibody to actin for protein loading control. (B) Flow cytometric analysis of cell cycle progression in unirradiated control and UV-treated cells, harvested 48 h after irradiation. (C) Percentage of apoptotic cells in unirradiated control samples (empty bars) and in cells treated with 10 J/m2 (solid bars) and re-incubated in complete medium for 24 h. Cells were judged to be apoptotic based upon morphological examination by fluorescence microscopy. Mean values ± SEM of at least three independent experiments are shown.
|
|
p53RPA interaction is lost after UV irradiation of IGROV-1, but not IGROV-1/Pt1 cells
In order to establish whether the early steps of the NER pathway could be affected by p53 sequestration by RPA, the modulation of the interaction between these two proteins was investigated in both cell lines. P53 protein was immunoprecipitated in cell extracts obtained from untreated controls, or from samples UV-irradiated and re-incubated for 3 h after damage. Figure 3A
shows the immunoblot analysis of RPA protein co-immunoprecipitated with p53 (IP
-p53), both in IGROV-1 and IGROV-1/Pt1 cells. Replication protein A, detected with an antibody against the 32 kDa subunit, was found in immunoprecipitates obtained from unirradiated control samples of both cell lines. However, after UV irradiation, disruption of the interaction could be observed in the parental IGROV-1 cells, but not in p53-mutant cells. Western blot analysis of the cell extracts before immunoprecipitation shows that in each cell line, the levels of RPA (32 kDa) protein were not significantly modified by the UV treatment (WB RPA). As expected, the levels of p53 protein before immunoprecipitation (WB p53) appear to be modulated in IGROV-1, but not in IGROV-1/Pt1 cells. To further assess whether other DNA damaging agents could induce a similar effect, IGROV-1 and IGROV-1/Pt1 cells were exposed for 1 h to 100 and 300 µg/ml cisplatin, respectively. Samples were then incubated in drug-free medium for 2 h, harvested and p53 immunoprecipitated as above. Similarly to UV radiation, cisplatin induced a decrease in the stability of the RPAp53 association in IGROV-1 cells, but not in IGROV-1/Pt1 cells (Figure 3B
).

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 3. Modulation of p53RPA interaction in IGROV-1 and IGROV-1/Pt1 cells after DNA damage. Cells were (A) exposed to UV-C radiation (10 J/m2) and incubated for 3 h, or (B) treated for 1 h with cisplatin followed by 2 h incubation in drug-free medium. IP -p53, Immunoblot analysis of RPA protein (32 kDa subunit) immunoprecipitated with p53 antibody in control and treated samples; WB, western blot analysis of RPA (32 kDa) and p53 proteins in whole cell extracts of the same samples before immunoprecipitation.
|
|
DNA incision efficiency
In the light of the above results, it was interesting to assess whether the first steps of NER, i.e. damage recognition and incision, were impaired by p53 mutation in IGROV-1/Pt1 cells, DNA incision efficiency was evaluated by the alkaline elution method. Samples were incubated in the presence of the DNA repair inhibitors araC and HU, in order to prevent DNA synthesis and subsequent rejoining after enzymatic incision (41). Elution profiles were obtained from untreated control cells and from samples irradiated with 10 J/m2 UV-C light and incubated for 5 min or 3 h after irradiation. As compared with the untreated control samples, the elution profiles of UV-irradiated cells were significantly modified indicating that DNA was already readily incised 5 min after irradiation (Figure 4A
). The samples incubated for 3 h after UV exposure showed similar elution curves (data not shown). The mean values of the rate constants calculated on the slope of the DNA elution profiles of each sample are reported in Figure 4B
. These values showed no significant differences between the two cell lines, indicating that the process was virtually complete in the early phase after damage. No determination of DNA incision at later times was attempted due to the significant levels of apoptosis-induced DNA strand breaks in IGROV-1 cells. By comparison with the samples exposed to
-rays, it was estimated that UV light induced DNA strand breaks at a frequency of ~45 DNA strand breaks/109 Da, i.e. in the same range of values obtained on other tumour cell lines (41).
Recruitment of PCNA to DNA repair sites
The ability of IGROV-1 and IGROV-1/Pt1 cells to recruit proteins necessary for DNA repair synthesis after the incision/excision step was evaluated by determining the recruitment of soluble PCNA to insoluble sites on chromatin. Figure 5A
shows the western blot analysis of PCNA into the soluble and detergent-insoluble fractions. At 30 min post-UV irradiation, an increase in the amount of chromatin-bound PCNA could be observed in both cell lines, while no modification was evident in the soluble fraction. Flow cytometric analysis of immunofluorescence enables the evaluation of the cell cycle distribution of the detergent-insoluble form of PCNA in untreated and UV-irradiated cells (Figure 5B
). In control samples, high levels of PCNA are normally present only in S-phase cells, where PCNA participates in DNA replication. In both cell lines, the recruitment of PCNA to the insoluble repair sites was readily detectable 30 min after irradiation in G1 and G2 + M phases. The kinetics of PCNA recruitment was also investigated at various time points after irradiation. Figure 6
shows the quantitative analysis of PCNA immunofluorescence measured in the G1 phase of irradiated samples and normalized to the values of untreated control cells. The results show that after an initial 3-fold increase in insoluble PCNA in UV-treated samples, a further increase (up to 45-fold) was detectable between 8 and 24 h post-irradiation. PCNA levels returned nearly to basal values at 48 h after DNA damage in IGROV-1 cells. In remarkable contrast, no significant reduction in chromatin-associated PCNA could be observed between 24 and 48 h in p53-mutant IGROV-1/Pt1 cells.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 5. Recruitment of PCNA to the insoluble sites of nuclear chromatin 30 min after UV-C irradiation of IGROV-1 and IGROV-1/Pt1 cells. (A) Western blot analysis of the detergent-soluble (S) and insoluble (I) forms of PCNA in untreated controls and in UV-irradiated samples. (B) Immunocytometric determination of detergent-insoluble PCNA in hypotonically lysed cells. The three-dimensional plots show the cell cycle distribution of immunofluorescence relative to insoluble PCNA in UV-irradiated and in untreated control cells.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 6. Time course of PCNA recruitment to repair sites after UV-irradiation (10 J/m2) of IGROV-1 and IGROV-1/Pt1 cells. Bars represent the amount of insoluble PCNA in the G1 phase of the cell cycle. Immunofluorescence signals were normalized to unirradiated control samples. Mean values ± SEM of three independent experiments are shown.
|
|
CPDs removal in UV-irradiated IGROV-1 and IGROV-1/Pt1 cells
Since the recruitment of PCNA after UV irradiation occurred to a similar extent in both cell lines, at least in the early steps of the repair process, the ability to remove the CPD lesions was assessed by an immunoblot method. The initial amount of damage, as determined by the band intensities of zero-time irradiated samples, was not significantly different in the two cell lines (Figure 7A
). Band quantification by densitometric analysis showed that the percentage of CPDs removed after 3 h incubation post-UV exposure (ranging from about 15 to 25%) was not significantly different in the two cell lines. In contrast, at 24 h after UV irradiation the extent of repair was ~50% of the initial amount of lesions in IGROV-1 cells. A remarkably lower level of repair (~25%) was observed for the p53-mutant IGROV-1/Pt1 cells (Figure 7B
).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 7. Analysis of CPD removal during DNA repair in IGROV-1 and IGROV-1/Pt1 cells irradiated with UV-C (10 J/m2). (A) Immunoblot analysis of CPDs in DNA extracted from control and UV-irradiated samples. (B) CPD removal assessed by densitometric analysis of the bands, as shown in (A). Mean values ± SEM of three independent experiments are shown.
|
|
 |
Discussion
|
---|
UV irradiation of IGROV-1 cells induced a normal p53 response, as indicated by modulation of p53 levels, cell cycle progression and occurrence of apoptosis. The absence of these events in IGROV-1/Pt1 cells confirmed the lack of transcriptional activity of the mutant form of p53, similar to what has previously been observed after DNA damage induced by ionizing radiation (40). The finding that IGROV-1/Pt1 cells were more resistant to UV than the p53-wild type parental cells cannot be explained by differences in mismatch repair (which is associated to cisplatin resistance) since both cell lines neither express MLH1, nor PMS2 proteins (P.Gatti and P.Perego, personal communication). Although it cannot be excluded that additional changes, other than p53 mutations, may have occurred in IGROV-1/Pt1 cells, karyotyping showed only a rearrangement of chromosome 4, in comparison with the parental cells (47). No changes in chromosomes carrying NER genes (24), nor differences in the expression of other relevant DNA replication/repair genes, like DNA topoisomerases I and II
and DNA polymerase ß, were found (47). The UV resistance of IGROV-1/Pt1 cells is consistent with that observed in LFS fibroblasts, carrying mutations within the DNA binding domain of p53 (11,48). In other systems in which DNA repair was affected by p53 inactivation with E6 protein (13), or by impairing downstream p53 effectors, like gadd45 and p21, a greater UV sensitivity was observed (49,50). In these systems, however, cells retained the ability to undergo apoptotic cell death, thus explaining the different survival after DNA damage.
Previous studies addressing the repair efficiency of tumour cells suggested that the lack of a functional p53 was important in terms of the interaction with proteins XPB and XPD, components of the TFIIH transcription complex (14). However, since p53 inhibits the activity of these proteins, it has been difficult to indicate a role for this interaction in NER. It was recently shown that the p53RPA interaction was disrupted after UV-induced DNA damage, thus suggesting that release of RPA and p53 could facilitate the respective function of each protein in NER (23). Interestingly, the association was not disrupted in cells defective in global genome repair (23). In agreement with these findings, IGROV-1/Pt1 cells, but not the parental cells, failed to down-regulate this interaction, thus suggesting that NER efficiency and p53 status play a role in modulating the stability of the RPAp53 interaction.
Down-regulation of RPAp53 interaction does not appear to be required for DNA incision since IGROV-1/Pt1 cells exhibited an amount of UV-induced DNA strand breaks similar to that measured in the parental cells. These findings also imply that recognition of the lesions in p53-mutant IGROV-1/Pt1 cells is normal, since in NER-deficient cell lines with mutation in genes involved in the recognition step (e.g. XP-A), DNA incision is drastically reduced (41). In previous studies, the amount of DNA strand breaks induced by UV light in LFS fibroblasts was lower than in normal cells (9). The discrepancy with our results may be related to the cell lines, in addition to the different techniques used for determining the amount of DNA breaks. In fact, the extent of DNA incision after UV exposure of p53-null Saos-2 cells was similar to that of a derivative cell clone expressing wild-type p53 (51).
DNA incision is known to be a rate limiting step for the PCNA-dependent DNA repair, as assessed in vitro with UV-damaged plasmids (24,25). Accordingly, XP-A and XP-G cells, belonging to complementation groups that are defective in DNA damage recognition, or in the DNA incision step, respectively, are unable to recruit PCNA in the insoluble chromatin fraction immediately after irradiation (52).
The recruitment of PCNA to DNA repair sites is a fast process (38), with maximal levels being reached in 1530 min after irradiation of normal fibroblasts (44,45). In IGROV-1/Pt1 and parental cells, such an event occurred with a similar initial kinetics and extent. These results indicate that neither DNA incision nor early recruitment of PCNA to DNA repair sites was affected by a mutant p53 protein. Remarkably, the persistent binding of PCNA to chromatin at late repair times occurred concomitantly with a reduction in the removal of CDPs in IGROV-1/Pt1, as compared with parental cells. A similar phenomenon has been recently observed in p21waf1/cip1-null human fibroblasts (50). Thus, absence of p21 protein (as due to gene deletion or lack of transcription) may result in the persistence of PCNA at damaged sites, thereby affecting DNA repair. Although the mechanism of this defect has not yet been elucidated, it might be related to the lack of removal/recycling of PCNA. In fact, p21 could be required in this process to complete late repair steps, such as DNA ligation and chromatin assembly (45,50). That high PCNA levels may be associated with NER deficiency is also suggested by evidence showing that both the increased expression of PCNA induced by the viral Tax protein and the direct overexpression of PCNA were able to affect NER efficiency (53). In this system, overexpression of p21, but not of a mutant form unable to interact with PCNA, rescued NER (54). In other studies, the expression of p21, and particularly its interaction with PCNA, was reported to be important for NER efficiency (55,56). This common feature suggests that the lack of transcriptional activation of the p21waf1/cip1 gene by p53 may induce some alteration in the repair pathway, leading to NER impairment. In fact, the suppression of DNA repair by the viral Tax protein was rescued by a functional p53 transactivating activity (54). Repair defects have been also described in cells lacking gadd45, another p53-downstream protein involved in NER (57). Similarly, the p48-XPE protein involved in the recognition of the damage is under transcriptional control of p53 (58). The mRNA and protein expression of p48 have been found to increase at a late stage after DNA damage (59), suggesting an important role for p53-mediated transcription in the late steps of NER. These findings outline the importance of the transactivation of relevant NER genes, rather than a direct role of p53 in the repair process.
In conclusion, disruption of the global NER pathway in ovarian carcinoma cells with mutant p53 may occur because of a lack of the required protein expression required in the late stages of the repair process.
 |
Notes
|
---|
3 To whom correspondence should be addressedEmail: prosperi{at}dragon.ian.pv.cnr.it 
 |
Acknowledgments
|
---|
We thank Prof. M.Wold for providing the anti-RPA antibody and R.Melli for help in computer drawings.
 |
References
|
---|
-
Hartwell,L.H. and Kastan,M.B. (1994) Cell cycle control and cancer. Science, 266, 18211828.[ISI][Medline]
-
Zhou,B.B. and Elledge,S.J. (2000) The DNA damage response: putting checkpoints in perspective. Nature, 408, 433439.[ISI][Medline]
-
Kemp,C.J., Wheldon,T. and Balmain,A. (1994) p53-deficient mice are extremely susceptible to radiation-induced tumorigenesis. Nature Genet., 8, 6670.[ISI][Medline]
-
Havre,P.A., Yuan,J.L., Hedrick,L., Cho,K.R. and Glazer,P.M. (1995) p53 inactivation by HPV16 E6 results in increased mutagenesis in human cells. Cancer Res., 55, 44204424.[Abstract]
-
Parshad,R., Price,F.M., Pirollo,K.F., Chang,E.H. and Sanford,K.K. (1993) Cytogenetic response to G2-phase X irradiation in relation to DNA repair and radiosensitivity in a cancer-prone family with LiFraumeni Syndrome. Radiat. Res., 136, 236240.[ISI][Medline]
-
Donner,E.M. and Preston,R.J. (1996) The relationship between p53 status, DNA repair and chromatid aberration induction in G2 mouse embryo fibroblast cells treated with bleomycin. Carcinogenesis, 17, 11611165.[Abstract]
-
Smith,M.L., Chen,I.T., Zhan,Q., O'Connor,P.M. and Fornace,A.J.Jr (1995) Involvement of the p53 tumour suppressor in repair of UV-type DNA damage. Oncogene, 10, 10531059.[ISI][Medline]
-
Rainbow,A.J. (1989) Defective repair of UV-damaged DNA in human tumor and SV-40 transformed human cells but not in adenovirus-transformed human cells. Carcinogenesis, 10, 10731077.[Abstract]
-
Mirzayans,R., Enns,L., Dietrich,K., Barley,R.D.C. and Paterson,M.C. (1996) Faulty DNA polymerase
/
-mediated excision repair in response to
-radiation or ultraviolet light in p53-deficient fibroblast strains from affected members of a cancer-prone family with LiFraumeni syndrome. Carcinogenesis, 17, 691698.[Abstract]
-
McKay,B.C., Francis,M.A. and Rainbow,A.J. (1997) Wild-type p53 is required for heat shock and ultraviolet light enhanced repair of a UV-damaged reporter gene. Carcinogenesis, 18, 245249.[Abstract]
-
Ford,J.M. and Hanawalt,P.C. (1995) LiFraumeni syndrome fibroblasts homozygous for p53 mutations are deficient in global DNA repair but exhibit normal transcription-coupled repair and enhanced UV resistance. Proc. Natl Acad. Sci. USA, 92, 88768880.[Abstract]
-
Ford,J.M. and Hanawalt,P.C. (1997) Expression of wild-type p53 is required for efficient global genomic nucleotide excision repair in UV-irradiated human fibroblasts. J. Biol. Chem., 272, 2807328080.[Abstract/Free Full Text]
-
Ford,J.M. and Hanawalt,P.C. (1998) Human fibroblasts expressing the human papillomavirus E6 gene are deficient in global genomic nucleotide excision repair and sensitive to ultraviolet irradiation. Cancer Res., 58, 599603.[Abstract]
-
Wang,X.W., Yeh,H., Schaeffer,L. et al. (1995) p53 modulation of TFIIH associated nucleotide excision repair activity. Nature Genet., 10, 188195.[ISI][Medline]
-
McKay,B., Winrow,C. and Rainbow,A.J. (1997) Capacity of UV-irradiated human fibroblasts to support adenovirus DNA synthesis correlates with transcription-coupled repair and is reduced in SV-40 transformed cells and cells expressing mutant p53. Photochem. Photobiol., 66, 659664.[ISI][Medline]
-
Abrahams,P.J., Houweling,A., Cornelissen-Steijger,P.D.M. et al. (1998) Impaired DNA repair capacity in skin fibroblasts from various hereditary cancer-prone syndromes. Mutat. Res., 407, 189201.[ISI][Medline]
-
Therrien,J.P., Drouin,R., Baril,C. and Drobetsky,E.A. (1999) Human cells compromised for p53 function exhibit defective global and transcription-coupled nucleotide excision repair, whereas cells compromised for Rb function are defective only in global repair. Proc. Natl Acad. Sci. USA, 96, 1503815043.[Abstract/Free Full Text]
-
Zhu,Q., Wani,M.A., El-mahdy,M. and Wani,A.A. (2000) Decreased DNA repair efficiency by loss or disruption of p53 function preferentially affects removal of cyclobutane pyrimidine dimers from non-transcribed strand and slow repair sites in transcribed strand. J. Biol. Chem., 275, 1149211497.[Abstract/Free Full Text]
-
McKay,B.C., Ljungman,M. and Rainbow,A.J. (1999) Potential role for p53 in nucleotide excision repair. Carcinogenesis, 20, 13891396.[Abstract/Free Full Text]
-
Zhou,J., Ahn,J., Wilson,S.H. and Prives,C. (2001) A role for p53 in base excision repair. EMBO J., 20, 914923.[Abstract/Free Full Text]
-
Offer,H., Milyavsky,M., Erez,N., Matas,D., Zurer,I., Harris,C.C. and Rotter,V. (2001) Structural and functional involvement of p53 in BER in vitro and in vivo. Oncogene, 20, 581589.[ISI][Medline]
-
Dutta,A., Ruppert,J.M., Aster,J.C. and Winchester,E. (1993) Inhibition of DNA replication factor RPA by p53. Nature, 365, 7982.[ISI][Medline]
-
Abramova,N.A., Russel,J., Botchan,M. and Li,R. (1997) Interaction between replication protein A and p53 is disrupted after UV damage in a DNA repair-dependent manner. Proc. Natl Acad. Sci. USA, 94, 71867191.[Abstract/Free Full Text]
-
Wood,R.D. (1996) DNA repair in eukaryotes. Annu. Rev. Biochem., 65, 135167.[ISI][Medline]
-
Sancar,A. (1996) DNA excision repair. Annu. Rev. Biochem., 65, 4381.[ISI][Medline]
-
De Laat,W.L., Jaspers,N.G.J. and Hoeijmakers,J.H.J. (1999) Molecular mechanism of nucleotide excision repair. Genes Dev., 13, 768785.[Free Full Text]
-
Wakasugi,M. and Sancar,A. (1999) Order of assembly of human DNA repair excision nuclease. J. Biol. Chem., 274, 1875918768.[Abstract/Free Full Text]
-
Aboussekhra,A., Biggerstaff,M., Shivji,M.K.K., Vilpo,J.A., Moncollin,V., Podust,V.N., Protic,M., Hübscher,U., Egly,J.-M. and Wood,R.D. (1995) Mammalian nucleotide excision repair reconstituted with purified components. Cell, 80, 859868.[ISI][Medline]
-
He,Z., Henricksen,L.A.L., Wold,M.S. and Ingles,C.J. (1995) RPA involvement in the damage-recognition and incision step of nucleotide excision repair. Nature, 374, 566569.[ISI][Medline]
-
De Laat,W.L., Appeldoorn,E., Sugasawa,K., Weterings,E., Jaspers,N.G.J. and Hoeijmakers,J.H.J. (1998) DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair. Genes Dev., 12, 25982609.[Abstract/Free Full Text]
-
Miura,M., Domon,M., Sasaki,T. and Takasaki,Y. (1992) Induction of proliferating cell nuclear antigen (PCNA) complex formation in quiescent fibroblasts from a xeroderma pigmentosum patient. J. Cell Physiol., 150, 370376.[ISI][Medline]
-
Aboussekhra,A. and Wood,R.D. (1995) Detection of nucleotide excision repair incisions in human fibroblasts by immunostaining for PCNA. Exp. Cell Res., 221, 326332.[ISI][Medline]
-
Miura,M., Nakamura,S., Sasaki,T., Takasaki,Y., Shiomi,T. and Yamaizumi,M. (1996) Roles of XPG and XPF/ERCC1 endonucleases in UV-induced immunostaining of PCNA in fibroblasts. Exp. Cell Res., 226, 126132.[ISI][Medline]
-
Shivji,M.K.K., Kenny,M.K. and Wood,R.D. (1992) Proliferating cell nuclear antigen is required for DNA excision repair. Cell, 69, 367374.[ISI][Medline]
-
Nichols,A.F. and Sancar,A. (1992) Purification of PCNA as a nucleotide excision repair protein. Nucleic Acids Res., 20, 35593564.
-
Prosperi,E. (1997) Multiple roles of proliferating cell nuclear antigen: DNA replication, repair and cell cycle control. Prog. Cell Cycle Res., 3, 193210.[Medline]
-
Jònsson,Z.O. and Hübscher,U. (1997) Proliferating cell nuclear antigen: more than a clamp for DNA polymerases. BioEssays, 19, 967975.[ISI][Medline]
-
Toschi,L. and Bravo,R. (1988) Changes in cyclin/proliferating cell nuclear antigen distribution during DNA repair synthesis. J. Cell Biol., 107, 16231628.[Abstract]
-
Righetti,S.C., Perego,P., Corna,P., Pierotti,M.A. and Zunino,F. (1999) Emergence of p53 mutant cisplatin-resistant in ovarian carcinoma cells following drug exposure: spontaneously mutant selection. Cell Growth Differ., 10, 473478.[Abstract/Free Full Text]
-
Perego,P., Giarola,M., Righetti,S.C., Supino,R., Caserini,C., Delia,D., Pierotti,M.A., Miyashita,T., Reed,J.C. and Zunino,F. (1996) Association between cisplatin resistance and mutation of p53 gene and reduced bax expression in ovarian carcinoma cell systems. Cancer Res., 56, 556562.[Abstract]
-
Squires,S., Johnson,R.T. and Collins,A.R.S. (1982) Initial rates of DNA incision in UV-irradiated human cells. Differences between normal, xeroderma pigmentosum and tumour cells. Mutat. Res., 95, 389404.[ISI][Medline]
-
Fornace,A.J.Jr, Kohn,K.W. and Kann,H.E.Jr (1976) DNA single-strand breaks during repair of UV damage in human fibroblasts and abnormalities of repair in xeroderma pigmentosum. Proc. Natl Acad. Sci. USA, 73, 3943.[Abstract]
-
Kohn,K.W., Ewig,R.A.G., Erickson,L.C. and Zwelling,L.A. (1981) Measurements of strand breaks and cross-links by alkaline elution. In Friedberg E.C. and Hanawalt,P.C. (eds) DNA Repair: a Laboratory Manual of Research Techniques, Vol. 1. Dekker, New York, pp. 379401.
-
Prosperi,E., Stivala,L.A., Sala,E., Scovassi,A.I. and Bianchi,L. (1993) Proliferating cell nuclear antigen complex-formation induced by ultraviolet irradiation in human quiescent fibroblasts as detected by immunostaining and flow cytometry. Exp. Cell Res., 205, 320325.[ISI][Medline]
-
Savio,M., Stivala,L.A., Scovassi,A.I., Bianchi,L. and Prosperi,E. (1996) p21waf1/cip1 protein associates with the detergent-insoluble form of PCNA concomitantly with disassembly of PCNA at nucleotide repair sites. Oncogene, 13, 15911598.[ISI][Medline]
-
Vink,A.A., Bergen Henegouwen,J.B.A., Nikaido,O., Baan,R.A. and Roza,L. (1994) Removal of UV-induced DNA lesions in mouse epidermis soon after irradiation. J. Photochem. Photobiol. B, 24, 2531.[ISI][Medline]
-
Perego,P., Romanelli,S., Carenini,N., Magnani,I., Leone,R., Bonetti,A., PaolicchiA. and Zunino,F. (1998) Ovarian cancer cisplatin-resistant cell lines: multiple changes including collateral sensitivity to taxol. Ann. Oncol., 9, 423430.[Abstract]
-
Barley,R.D.C., Enns,L., Paterson,M.C. and Mirzayans,R. (1998) Aberrant p21WAF1-dependent growth arrest as the possible mechanism of abnormal resistance to ultraviolet light cytotoxicity in LiFraumeni syndrome fibroblast strains heterozygous for TP53 mutations. Oncogene, 17, 533543.[ISI][Medline]
-
Smith,M.L., Kontny,H.U., Zhan,Q., Sreenath,A., O'Connor,P.M. and Fornace,A.J.Jr (1996) Antisense GADD45 expression results in decreased DNA repair and sensitizes cells to UV-irradiation or cisplatin. Oncogene, 13, 22552263.[ISI][Medline]
-
Stivala,L.A., Riva,F., Cazzalini,O., Savio,M. and Prosperi,E. (2001) p21waf1/cip1-null human fibroblasts are deficient in nucleotide excision repair downstream the recruitment of PCNA to DNA repair sites. Oncogene, 20, 563570.[ISI][Medline]
-
Yagi,T., Mohri-Nakanishi,K., Matsuda,T., Yamagishi,N., Miyakoshi,J. and Takebe,H. (1998) Reduced UV-induced mutations in human osteosarcoma cells stably expressing transfected wild type p53 cDNA. Cancer Lett., 123, 7176.[ISI][Medline]
-
Miura,M. (1999) Detection of chromatin-bound PCNA in mammalian cells and its use to study DNA excision repair. J. Radiat. Res., 40, 112.
-
Kao,S.Y. and Mariott,S.J. (1999) Disruption of nucleotide excision repair by the human T-cell leukemia virus type 1 Tax protein. J. Virol., 73, 42994304.[Abstract/Free Full Text]
-
Kao,S.Y., Lemoine,F.J. and Marriott,S.J. (2000) Suppression of DNA repair by human T cell leukemia virus type 1 Tax is rescued by a functional p53 signaling pathway. J. Biol. Chem., 275, 3592635931.[Abstract/Free Full Text]
-
McDonald III,E.R., Wu,G.S., Waldman,T. and El-Deiry,W.S. (1996) Repair defect in p21WAF1/CIP1 / human cancer cells. Cancer Res., 56, 22502255.[Abstract]
-
Sheikh,M.S., Chen,Y.Q., Smith,M.L. and Fornace,A.J.Jr. (1997) Role of p21waf1/cip1/sdi1 in cell death and DNA repair as studied using a tetracycline-inducible system in p53-deficient cells. Oncogene, 14, 18751882.[ISI][Medline]
-
Smith,M.L., Ford,J.M., Hollander,C., Bortnick,R.A., Amundson,S.A., Seo,Y.R., Deng,C.-X., Hanawalt,P.C. and Fornace,A.J.Jr (2000) p53-mediated DNA repair responses to UV radiation: studies of mouse cells lacking p53, p21 and/or gadd45 genes. Mol. Cell Biol., 20, 37053714.[Abstract/Free Full Text]
-
Hwang,B.J., Ford,J.M., Hanawalt,P.C. and Chu,G. (1999) Expression of the p48 xeroderma pigmentosum gene is p53-dependent and is involved in global genome repair. Proc. Natl Acad. Sci. USA, 96, 424428.[Abstract/Free Full Text]
-
Nichols,A.F., Itoh,T., Graham,J.A., Liu,W., Yamaizumi,M. and Linn,S. (2000) Human damage-specific DNA-binding protein p48. Characterization of XPE mutations and regulation following UV irradiation. J. Biol. Chem., 275, 2142221428.[Abstract/Free Full Text]
Received May 11, 2001;
revised August 15, 2001;
accepted September 5, 2001.