Guy-Bernier Research Center, Maisonneuve-Rosemont Hospital, Faculty of Medicine, University of Montreal, 5415 boulevard de lAssomption, Montréal, Québec, Canada, H1T 2M4
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Abstract |
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Abbreviations: CPD, cyclobutane pyrimidine dimers; GNER, global nucleotide excision repair; IR, ionizing radiation; NER, nucleotide excision repair; PI, propidium iodide; SSL, simulated sunlight; 6-tg, 6-thioguanine.
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Introduction |
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Besides through loss of apoptotic capacity, inactivation of p53 would also be expected to favor photocarcinogenesis through a reduction in cellular DNA repair efficiency. Indeed, different lines of evidence demonstrate that p53 regulates nucleotide excision repair (NER) (68), a critical antineoplastic pathway that is essential for removing highly genotoxic helix-distorting DNA lesions, including UV-induced cyclobutane pyrimidine dimers (CPD). NER is comprised of two overlapping subpathways that differ only in the initial (lesion-recognition) step: (i) global NER (GNER), which removes DNA adducts from virtually anywhere in the genome and (ii) transcription-coupled NER (TCNER) that accomplishes more rapid repair of lesions located uniquely on the transcribed strand of active genes (9). Despite the general accord that functional p53 is essential for efficient NER, it remains controversial whether this tumor suppressor regulates GNER only versus both GNER and TCNER (see Discussion).
Following exposure to DNA damaging agents, the G1 cell-cycle checkpoint apparently allows more time for DNA repair prior to S phase, thereby forestalling the replication of damaged DNA templates and protecting against mutagenesis and carcinogenesis. In the case of cells exposed to IR, the critical involvement of p53 in G1 arrest, i.e. via transcriptional upregulation of the cyclin-dependent kinase inhibitor p21waf1 and consequent dephosphorylation of the retinoblastoma tumor suppressor, has been unequivocally established (1012). However, it is now becoming increasingly evident that the initiation of G1 arrest may not depend on the p53/p21waf1 pathway in 254 nm UV-exposed human cells (13).
The capacity of various high-risk human papillomavirus (HPV) subtypes to promote anogenital and other squamous cell carcinomas is exerted largely through intracellular expression of the HPV-encoded E6 oncoprotein (14). E6 expression strongly stimulates the ubiquitin-mediated proteasomal degradation of p53, thereby efficiently inhibiting the latters ability to accumulate and transactivate downstream effectors (15). Furthermore, exogenous expression of E6 in cultured cells leads to clear manifestations of defective p53-dependent DNA damage processing, including abrogation of cell-cycle checkpoints and apoptosis (16), as well as reduced NER capacity (7,17). As such, E6 expression has been extensively employed as a model for characterizing the influence of p53 inactivation on the cellular response to DNA damage. However, E6 has also been shown to modulate the activity of a plethora of proteins aside from p53 (18), and indeed, the extent to which intracellular expression of this viral oncoprotein reflects a true p53-null phenotype following mutagen exposure is still not clear.
To further elucidate the influence of genetic-versus E6-mediated inactivation of p53 in the cellular response to UV, we employed the p53-proficient human lymphoblastoid strain TK6 (19), as well as two isogenic derivatives, i.e. TK6-5E wherein p53 protein is functionally inactivated via intracellular E6 oncoprotein expression (20), and NH32 which carries a homozygous knockout of the p53 gene (21). The TK6 lymphoblastoid system was chosen due to its status as an extremely well-characterized human cultured cell model for probing the response to genotoxic agents, and the fundamental role of p53 in this process. In addition, we highlight recent evidence demonstrating that UV exposure can strongly induce lymphoid tumors in mice in a p53-dependent manner (22). Among the three TK6-derived strains, cytotoxicity, mutagenesis, apoptosis and cell-cycle arrest were each quantitatively compared following irradiation with different UV wavelengths, i.e. the model mutagen 254 nm UV, as well as broad-spectrum simulated sunlight (SSL) which is designed to mimic terrestrial solar light (composed of UVB + UVA + visible light, and virtually devoid of wavelengths below 290 nm). In addition, to qualitatively evaluate the role of p53 in UV mutagenesis, we determined the DNA sequence specificity of SSL-induced mutations at the chromosomal hypoxanthine phosphoribosyltransferase (hprt) locus in each TK6-derived strain. This detailed comparison in a model isogenic system has permitted a rigorous analysis of p53-dependent DNA damage processing in UV-exposed human cells, as well as direct assessment of the general relevance of E6 expression as a paradigm for investigating the phenotypic consequences of p53 inactivation.
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Materials and methods |
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Irradiation conditions
Prior to irradiation with 254 nm UV, SSL or IR, exponentially growing cells were washed with Dulbeccos phosphate buffered saline (PBS), resuspended in 5 ml PBS, and added to 100 mm culture dishes on ice. Cells were treated with 254 nm UV light using a Philips G25T8 germicidal lamp (Philips, Eindhoven, The Netherlands), at a fluence of 0.2 Jm-2 s-1 as measured by a DRC 100x digital radiometer (Spectroline, Westbury, NY) In the case of IR, cells were exposed to a cesium137 source (Gamma Cell; Atomic Energy Canada, Ottawa, Canada) at a dose rate of 6.3 rad/s. For SSL exposure, cells were irradiated using a solar simulator equipped with a 2500 W xenon compact arc lamp (Conrad-Hanovia, Newark, NJ), at a fluence of 2 kJm-2 s-1according to a YSI-Kettering 65A radiometer (Yellow Springs Instruments, OH). The spectral characteristics and genotoxic effects of the SSL source employed here have been described in detail previously (23). Briefly, the incident SSL was rigorously purified using 3 mm thick glass filters (type WG 320; Schott, Mainz, Germany) in order to virtually eliminate contaminating wavelengths from the UVC region (i.e. below 290 nm). As estimated using the filter transmission and solar lamp spectral profiles supplied by the manufacturers, the incident SSL was comprised of <10-7% UVC, 0.8% UVB, 6% UVA, 43% visible light and 47% infrared. These same proportions for terrestrial sunlight at sea level are approximately <10-7, 0.3, 5, 62 and 32%, respectively (24).
Western blotting
Cells (2x106) were added to 60 mm dishes and immediately irradiated (or mock-irradiated) with 150 rad of IR. Following incubation for 3 h, cells were extracted in radioimmunoprecipitation assay buffer [10 mM TrisHCl pH 8, 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40 and 1% sodium deoxycholate], containing 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM phenylmethylsulphonyl fluoride and 1 µM Na3VO4, followed by sonication, and clarification by centrifugation. Protein content was determined using the bicinchoninate assay (Pierce, Rockford, IL) according to manufacturers specifications. Aliquots containing 50 µg of protein were resolved on 7.5% acrylamide gels and electroblotted onto nitrocellulose membranes. The membranes were blocked in 5% (w/v) non-fat dry milk/PBS containing 0.1% (w/v) sodium azide, and incubated overnight at 4°C with primary antibodies against p53 (DO-1, Santa Cruz Biotechnology, Santa Cruz, CA) or p21 (Ab-1, Calbiochem, San Diego, CA), diluted 1:5000 and 1:800, respectively, in 1% (w/v) milk/PBS. The proteins were detected by chemiluminescence using a secondary antimouse-antibody coupled to horseradish peroxidase (Amersham, UK).
Clonogenic survival
Clonogenic survival was determined according to DeLuca et al. (25). Immediately following irradiation with 254 nm UV, SSL or IR, cells were collected by centrifugation and resuspended in normal growth medium, followed by dilution and plating of appropriate cell numbers in replicate 96 well dishes. After 2 weeks incubation, dishes were scored for colony formation. Clonogenic survival was calculated on the basis of a Poisson distribution. Results are expressed as the average ± SEM of at least four independent experiments.
Apoptosis analysis
Apoptosis was measured by double staining with Hoechst 33342 and propidium iodide (PI; Molecular Probes, Eugene, OC) as described previously for strain TK6 (26). Briefly, 2x106 cells were irradiated in exponential growth phase with equitoxic doses of 254 nm UV, SSL or IR and incubated in regular growth medium for 24, 48, 72 or 96 h. Cells (5x105) were then thoroughly washed with PBS and incubated for 15 min at 37°C in the dark in 100 µl of PBS + 1 µg/ml Hoescht 33342. The cells were centrifuged and resuspended in 1 ml of PBS containing 5 µg/ml of PI and 4 µg/ml of RNase A, and immediately analysed by fluorescence activated cell sorting using a FACStar apparatus (Becton-Dickinson, Franklin Lakes, NJ) equipped with a helium/cadmium laser emitting at 325 nm. Each time point represents the average ± SEM of at least four independent experiments.
Cell-cycle analysis
Cells (2 x 106) were irradiated with equitoxic doses of 254 nm UV, SSL or IR during exponential growth, followed immediately by addition of the mitotic inhibitor colcemid (1 µM final concentration) to block the re-entry of cells into G1. At various time points following addition of colcemid, irradiated or mock-treated cells were washed with PBS containing 50 mM EDTA, resuspended in 1 ml of PBS/EDTA, and then fixed by addition of 3 ml ice-cold 100% ethanol. Cells were then pelleted, washed with 4 ml of PBS/EDTA, and stained with modified Krishan buffer [0.05 mg/ml PI, 0.1% sodium citrate, 0.2 mg/ml RNase A and 0.3% (v/v) NP-40]. The fraction of the population in each phase of the cell cycle was then determined as a function of DNA content using a FACScan flow cytometer equipped with CellFit software (Beckton Dickinson). The delay in G1S progression is expressed as the percentage of cells remaining in the G0G1 compartment over time. Each time point represents the average ± SEM of at least three independent experiments.
Mutation frequency at the chromosomal hprt locus
Bulk cultures of TK6, NH32 or TK6-5E were grown for 2 days in RPMI medium supplemented with CHAT (2 x 10-5 M cytidine, 2 x 10-4 M hypoxanthine, 2 x 10-7 M aminopterin and 2 x 10-5 M thymidine) in order to reduce the background level of hprt- mutants. CHAT-treated strains were then propagated for 3 days in regular medium, after which appropriate numbers of cells were added to 100 mm dishes and irradiated with various doses of 254 nm UV, SSL or IR. At least 2x106 surviving cells from treated cultures were maintained for 7 days in normal RPMI medium to allow expression of the hprt- phenotype. To select for hprt- mutants, at least 7.5 x 106 treated cells were then seeded in 96 well dishes (40 000 cells/well) in RPMI medium supplemented with the purine analog 6-thioguanine (6-tg) at concentrations of 0.9 µg/m for TK6 and TK6-5E, and 10 µg/m for NH32 (see Results section for explanation of this 6-tg dose differential). Dishes were incubated for 21 days and scored for colony formation. Mutant frequencies were calculated according to a Poisson distribution as described previously (27). Results are expressed as the average ± SEM of at least three independent experiments.
DNA sequence characterization of SSL-induced hprt- mutants
A total of 49, 40 and 52 independent hprt- mutants for TK6, NH32, TK6-5E, respectively, were isolated following exposure to 500 kJm-2 of SSL, and characterized at the cDNA sequence level. For each mutant, total RNA was extracted from 5x105 cells using TRIzol reagent (Gibco BRL), and quantified by spectrophotometry. Two micrograms of RNA were then used as template for reverse transcription in a reaction mixture containing 50 mM TrisHCl, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 500 µM of each dNTP, 0.1 µg/µl BSA, 10 ng/µl of oligo d(T)1218, 1 U/µl of RNAsin and 2.5 U/µl of M-MuLV reverse transcriptase. After 1 h incubation at 37°C, the following reagents were added directly to the reaction tube (total final volume 50 µl): 400 µM of each dNTP (Gibco BRL), 0.5 ng/µl of primers PCR-1 and PCR-4 (sequence is given below), 2.5 U Taq polymerase (Gibco BRL), and 5 µl of 10x PCR buffer. This mixture was submitted to the following PCR reaction: 94°C (1 min), 55°C (1 min) and 72°C (2 min) for 30 cycles, followed by one cycle of 72°C (7 min). From this first PCR reaction, 1 µl was then added to the following reaction mix (final volume 50 µl): 2 µl of each dNTP (10 mM stock), 5 µl of PCR buffer 10x, 2.5 U Taq polymerase (Gibco BRL), and 2.5 µl of primers PCR-2 and PCR-3 (1.0 OD stock concentration). This mix was submitted to the following PCR reaction: 94°C (1 min), 55°C (2 min) and 72°C (2 min) for 30 cycles. Following the above nested PCR amplification procedure, aliquots were visualized by agarose gel electrophoresis to ensure the presence of an hprt cDNA. Dideoxy sequencing of each mutant cDNA was performed using a Taq polymerase-based cycle sequencing protocol using the primers below.
Primers for RT-PCR.
PCR-1:5'-CTGCTCCGCCACCGGCTTCC-3'; PCR-2: 5'-GATAATTTTA- CTGGCGATGT-3'; PCR-3: 5'-CCTGAGCAGTCAGCCCGCGC-3'; PCR-4: 5'-CAA TAGGACTCCAGATGTTT-3'.
Primers for dideoxy sequencing.
hhSEQ-1:5'-CTATCACTATTTCTATTCAGTG-3'; hhSEQ-2: 5'-AAGGAGATGGGAGGCCATC-3'; hhSEQ-3: 5'-GTGGAAGATATAATTGACACTGG-3'; hhSEQ-4:5'-GGATTATACTGCCTGACCAAG-3'.
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Results |
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Clonogenic survival in p53-proficient versus -deficient human lymphoblastoid strains
To assess the influence of p53 on the cytotoxic effects of UV, we evaluated clonogenic survival for each of TK6, NH32 and TK6-5E after irradiation with approximately equitoxic dose ranges of SSL (0750 kJm-2), 254 nm UV (07.5 Jm-2) or IR (0400 rads). We note that IR was included as a control for most of the experiments in the current study, as this model mutagen has already been extensively employed for studies of p53-dependent DNA damage processing in the TK6 lymphoblastoid system (see references throughout). In the case of IR treatment, there was no difference in survival between TK6, NH32 and TK6-5E (Figure 2A), in accord with previous studies on these strains (20,21). Following exposure to either 254 nm UV or SSL, wild-type TK6 and p53-null NH32 again displayed virtually identical levels of cellular resistance; however, E6-expressing TK6-5E was moderately but significantly hypersensitive (Figure 2B and C
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DNA sequence specificity of mutations induced by SSL at the endogenous hprt locus in p53-proficient versus -deficient human lymphoblastoid strains
To determine whether inactivation of p53 can qualitatively modulate UV mutagenesis at a chromosomal locus in human cells, we irradiated TK6, NH32 and TK6-5E with 500 kJm-2 of SSL, followed by isolation and cDNA sequence level characterization of at least 40 independent SSL-induced hprt- mutants from each strain. For all three strains, exposure to this SSL dose resulted in at least 20% relative clonogenic survival (Figure 2C), as well as a significant (minimum 9-fold) induction in the frequency of hprt- mutations over spontaneous background levels (Figure 5C
). The spectra of SSL-induced base substitutions at the hprt locus in TK6, TK6-5E and NH32 is presented in Figure 6
. Over 90% of SSL-induced hprt- mutations in each strain were single base substitutions leading to either amino acid changes in the hprt protein, or to precise skipping of one or more exons during hprt mRNA processing. Moreover, in the case of each strain, the vast majority of SSL-induced base substitutions were targeted to potential dipyrimidine sites, strongly indicating that these mutations were caused via misreplication of unrepaired solar UV-induced CPDs.
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Discussion |
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While prior investigations on the role of p53 in the UV response have focused only on monochromatic 254 nm UV exposure, the present study also employed broad-spectrum SSL. It is of interest to compare the effects of both wavelength regions. Although not contained within the terrestrial solar wavelength spectrum, 254 nm UV is a model mutagen for which a huge genotoxic database already exists, while SSL mimics environmentally relevant sunlight but is much less well studied. Furthermore, these wavelength regions are known to exert different biological effects. Indeed, 254 nm UV does not significantly alter the redox state of the cell, being strongly absorbed specifically by DNA wherein it induces almost exclusively dipyrimidine-type photoproducts. In contrast, the environmentally relevant UVB and UVA wavelengths present in SSL are absorbed by a plethora of cellular macromolecules in addition to DNA, generating reactive oxygen species which in turn produce specific patterns of gene activation and diverse types of damage which differentially impact cellular responses relative to 254 nm UV (30). Our result showing that the vast majority of SSL-induced mutations in TK6 and NH32 are located at dipyrimidine target sites is consistent with the now well-established fact that broad-spectrum sunlight, like 254 nm UV, exerts its primary premutagenic potential through the induction of dipyrimidine-type photoproducts (mostly CPD), rather than of oxidative DNA damage in human cells (2,23,3133). This result also suggests that p53 does not sensitize cells to oxidative DNA damage, as might be postulated based on recent studies demonstrating a role for p53 in base excision repair (34), which removes highly mutagenic oxidized DNA bases such as 8-oxo-guanine that are known to be generated by solar wavelengths (30). Finally, as mentioned in more detail below, equitoxic doses of 254 nm UV and SSL induced very similar responses overall at the level of clonogenic survival, mutagenesis, apoptosis and cell-cycle arrest in both TK6 and NH32. This similarity between 254 nm UV and SSL indicates that the vast database on the cellular effects of the model mutagen 254 nm UV can be extrapolated with more confidence to the situation for environmentally relevant sunlight.
We demonstrate here that genetic inactivation of p53 in human lymphoblastoid cells does not affect cellular resistance to UV-induced cell killing, as judged by the virtually identical levels of clonogenic survival observed for TK6 versus NH32 following exposure to either 254 nm UV or SSL. Prior investigations in other human systems have yielded conflicting results regarding the influence of p53 inactivation on UV-induced cell death, e.g. adenocarcinoma cells transduced with dominant negative p53 manifested decreased clonogenic survival after UV exposure (35), whereas expression of wild-type p53 in p53-null osteosarcoma cells had no affect (36). The situation is further complicated by contradictory studies, each employing precisely the same p53-null skin fibroblast strains, which claimed that abrogation of p53 reduces (37) or increases (6) clonogenic survival after UV treatment. These latter investigations were able to correlate these cytotoxicity results with further conflicting data showing that wild-type p53 in their hands, respectively, inhibits or promotes UV-induced apoptosis. In any case, the demonstration here of profound resistance to programmed cell death exhibited by NH32 relative to TK6 after UV (or IR) exposure does not appear consistent with the observed virtually identical levels of clonogenic survival for these strains. However, as noted earlier, besides regulating apoptosis, functional p53 is also known to be required for NER following UV irradiation. Furthermore, while loss of apoptotic capacity greatly enhances clonogenic survival, loss of NER engenders precisely the opposite effect. Thus, it is plausible that for NH32, following UV exposure, any potential enhancement of clonogenic survival conferred by abrogation of p53-dependent apoptosis could be offset by an equivalent reduction in viability attributable to loss of the p53-dependent ability to remove highly genotoxic CPD via NER.
As growth arrest during G1 phase of the cell cycle is presumed to mitigate the mutagenic and carcinogenic effects of DNA damaging agents, it was important to investigate the potential role of p53 in UV-induced G1 arrest in our model system. Moreover, further characterization specifically of TK6 with respect to G1S progression after mutagen exposure may be of interest, since a previous report claimed that this strain atypically lacks the IR-induced G1 checkpoint, despite its p53-proficient status (38). Nonetheless, we found that while TK6 does not exhibit any arrest in G1S progression within the first 69 h following IR exposure (in agreement with the immediately aforementioned study), evaluation of later time points shows that this strain does in fact undergo a significant and sustained G1 arrest beginning at 9 h and continuing beyond 24 h post IR treatment. This growth delay was completely abrogated in NH32, which is expected given the preeminent role of p53 in IR-induced G1 arrest. In contrast to the situation for IR, however, NH32 manifested identical rates of S-phase entry compared with TK6 after exposure to either 254 nm UV or SSL. This reveals the presence of a potent p53-independent G1 arrest in UV-exposed human lymphoblastoid cells, which is in accord with recent studies in other human cell types irradiated with 254 nm UV (13,39). Therefore, if the existence of a DNA damage-inducible G1 checkpoint is important in guarding against neoplastic transformation, our results indicate that p53 inactivation, in the case of solar UV-induced skin cancer (or possibly lymphoma), does not impact such a protective process.
A previous investigation on NH32 revealed, relative to TK6, no change in mutation frequency at the endogenous thymidine kinase (tk) locus following treatment with IR (21). While we were able to reproduce this latter result on IR-induced mutagenesis at tk (data not shown), we nonetheless found that NH32 manifested slightly but significantly lower hprt mutation frequencies compared with TK6 after exposure to IR, as well as to either 254 nm UV or SSL. This may appear surprising, as loss of both apoptotic and NER capacities would be expected to enhance the accumulation of genetic damage within a given cell population, and hence to higher mutation frequencies in NH32 compared with TK6. Furthermore, our data differ from studies in other human tumor cell types which show that p53 inactivation can slightly enhance the frequency of 254 nm UV-induced hprt mutations (36,40). Other investigations using shuttle-vector carried supF targets in 254 nm UV-exposed murine cells have shown that abrogation of p53 activity can either increase (41) or have no effect (42,43) on mutation induction. At present we have no explanation for the slightly lower IR- and UV-induced hprt mutation frequencies in TK6 versus NH32. However, the fact that these strains display identical frequencies of IR-induced mutagenesis at tk suggests that our results for hprt may merely represent a locus-specific effect.
To assess any potential qualitative effects of genetic p53 inactivation on UV mutagenesis, we determined the DNA sequence specificity of SSL-induced mutations at the hprt locus in TK6 and NH32. To our knowledge, this represents the first DNA sequence-level characterization of mutations induced by any mutagen at an endogenous locus in isogenic human strains differing in p53 status. We found that NH32 manifested important disparities relative to TK6 in the proportion of SSL-induced hprt mutational classes, including a significant reduction in the frequency of base substitutions at A:T base pairs and a concomitant increase in exon-skipping events. Moreover, while some sites along the hprt locus were frequently mutated in both NH32 and TK6, it is intriguing that one particular G:C tract constituted a striking mutational hotspot only in NH32 TK6-SE, and is therefore specific for a p53-deficient background. Prior studies which analyzed shuttle-vector carried target genes after 254 nm UV treatment also revealed some differences between p53-proficient and -deficient murine cells in the distribution of mutational hotspots but, unlike the situation here, strong similarities in the relative proportion of mutational classes (42,43). One investigation in 254 nm UV-exposed human (osteosarcoma) cells, using essentially the same shuttle-vector system as the above murine studies, showed that p53 expression can significantly reduce the frequency of G:C to A:T transitions, as well as influencing the distribution of mutational hotspots (36). In any case, our own data strongly indicate that p53 status can be an important determinant in the probability of mutation fixation at certain, but not all, nucleotide positions at an endogenous locus in human cells. We speculate that this may occur through p53-mediated regulation of translesion bypass and/or of nucleotide excision repair rates, which might be expected to impact both the type and frequency of UV-induced mutations on a site-by-site basis.
As mentioned earlier, although it has been clearly shown that inactivation of p53 significantly compromises the efficiency of CPD removal via NER in human cells, conflicting data have emerged that directly demonstrate a role for p53 in GNER only (6,17) versus in both GNER and TCNER (7). The results presented here on the mutagenic specificity of SSL at an endogenous locus in isogenic human cells differing in p53 status shed some light on this controversy. We note that rodent cells lacking the murine homolog of human p48 (XP-E gene product) are deficient only in GNER, and manifest a highly significant preference for UV-induced hprt- mutations attributable to CPD originating on the non-transcribed strand (44). Similarly, in the case of humans, mutated p53 alleles in sunlight-induced skin tumors from XP-C patients, who are deficient in GNER only, show an extreme (100%) bias towards mutations due to unrepaired CPD on the non-transcribed strand (45,46). Thus, if p53 inactivation were to, e.g. profoundly reduce GNER efficiency but have no effect on TCNER, then one would clearly expect to recover a higher proportion of SSL-induced mutations due to unrepaired CPD on the non-transcribed strand of hprt (which is an actively transcribed locus) in NH32 relative to TK6. However, the two strains actually manifest a similar bias towards SSL-induced mutations on the transcribed strand. On this basis, our data are consistent with the notion that p53 regulates both GNER and TCNER.
The efficiency with which E6-expression is able to abrogate p53 function in diverse human and murine cell types has provided great impetus for its widespread use as a model to investigate the role of p53 in the DNA damage response. We were able to directly assess the validity of this model by comparing p53-null NH32 with TK6-5E, i.e. an isogenic counterpart that constitutively expresses the E6 oncoprotein and which is demonstrably deficient in major aspects of p53 function. We observed strong quantitative and qualitative correspondence between NH32 and TK6-5E with respect to mutagenesis at the endogenous hprt locus. Despite these similarities, however, following exposure to either 254 nm UV or SSL, relative to wild-type TK6, E6-expressing TK6-5E responded somehow differently than NH32 in virtually every other respect. Indeed, unlike NH32, TK6-5E displayed modest but significant hypersensitivity to the cytotoxic effects of either 254 nm UV or SSL, in agreement with previous studies showing that E6-expression can reduce clonogenic survival in different cultured cell types after UV exposure (35,40,47). Moreover, the apoptotic responses displayed by TK6-5E after treatment with either 254 nm UV or SSL were strikingly different, and more complex, than for p53-null NH32. Finally, although TK6-5E, like NH32, manifested a p53-independent G1S delay after treatment with 254 nm UV or SSL, this arrest endured for a considerably longer time period in the case of TK6-5E.
Although it is often taken for granted that intracellular E6 expression reflects a true p53-null phenotype, the major caveat nonetheless exists that this oncoprotein binds and interferes with the activity of many cellular proteins aside from p53. Furthermore, some of these E6-binding proteins are demonstrably or potentially implicated in the cellular response to genotoxic stress, e.g. the bcl-2 family member Bak (48), the (proapoptotic) c-myc transcription factor (49) and the transcriptional co-activator p300/CPB (50). Our own data comparing p53-null NH32 versus its isogenic E6-expressing counterpart TK6-5E provide a direct physiological demonstration that intracellular E6 expression can influence the global DNA damage response through p53-independent pathways. Furthermore, as demonstrated in the current study (Figure 1), intracellular E6 expression may severely attenuate, but not necessarily abrogate, levels of p53 protein. Therefore it cannot be ruled out that very low residual p53 activity in E6-expressing cells may influence certain p53-regulated responses to DNA damage. In any case, our results clearly emphasize the need for great caution when considering the use of intracellular E6 expression as an experimental paradigm for p53 inactivation.
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Acknowledgments |
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References |
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