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[Cancer Research 61, 3781-3786, May 1, 2001]
© 2001 American Association for Cancer Research

Regular Articles

Ablation of p21waf1cip1 Expression Enhances the Capacity of p53-deficient Human Tumor Cells to Repair UVB-induced DNA Damage1

Jean-Philippe Therrien, Martin Loignon, Régen Drouin and Elliot A. Drobetsky2

Division of Pathology, Department of Medical Biology, Faculty of Medicine, Laval University and Unité de Recherche en Génétique Humaine et Moléculaire, Research Centre, Hôpital St-François d’Assise, Centre Hospitalier Universitaire de Québec, Québec, Québec, Canada G1L 3L5 [J-P. T., R. D.], and Department of Microbiology and Immunology, Faculty of Medicine, Université de Montréal and Centre de Recherche Guy-Bernier, Hôpital Maisonneuve-Rosemont, Montréal [M. L., E. A. D.], Québec, Canada H1T 2M4


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
During periods of genotoxic stress, the cyclin-dependent kinase inhibitor p21waf1cip1 (hereafter referred to as p21) is transcriptionally up-regulated by the p53 tumor suppressor and subsequently plays a key role in cellular growth arrest. Investigations have also indicated that p21 may regulate nucleotide excision repair, a critical pathway that removes carcinogenic DNA damage induced by UV light and other mutagens. In this study, we examined whether low levels of endogenous p21 expression can modulate nucleotide excision repair in p53-deficient human tumor cells after UVB exposure. For this purpose, we used the well-characterized p53-/-p21+/+ adenocarcinoma cell strain DLD1 and its isogenic counterpart carrying a homozygous knockout for p21 (p53-/-p21-/- DLD1). Because p53-/-p21+/+ DLD1 expresses very low levels of endogenous p21 protein that are not up-regulated after mutagen exposure, this strain has been considered functionally p21-deficient in the cellular response to DNA damage. Nonetheless, the ligation-mediated PCR technology was used here to demonstrate, at nucleotide resolution, that p53-/-p21+/+ DLD1 excises UVB-induced cyclobutane pyrimidine dimers from the c-jun proto-oncogene at a significantly lower rate than the isogenic p53-/-p21-/- derivative. The higher efficiency of DNA repair in UVB-exposed p53-/-p21-/- DLD1 cells is accompanied by increased clonogenic survival and reduced levels of apoptosis, relative to the p53-/-p21+/+ counterpart. Our results show that ablation of p21 expression can significantly enhance the capacity of p53-deficient human tumor cells to repair UVB-induced DNA damage.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
NER3 is a critical antineoplastic pathway that removes helix-distorting DNA lesions generated by diverse mutagenic agents. These include highly genotoxic UVB-induced CPDs, which play a major role in the molecular etiology of sunlight-associated skin cancer (1 , 2) . Humans afflicted with the autosomal recessive disease xeroderma pigmentosum are defective in NER of UVB-induced CPD and manifest extreme sensitivity to cutaneous tumorigenesis (3) . The NER process is comprised, sequentially, of: (a) DNA damage recognition, followed by incision in a number of nucleotides on either side of the damaged base; (b) excision of the damaged base as part of an oligonucleotide approximately 30 nucleotides in length; and, finally (c) DNA resynthesis (gap-filling) and ligation, using normal DNA replication factors and the intact daughter strand as template. Two overlapping NER subpathways have been identified that essentially differ only in the initial (lesion recognition) step, i.e., GNER, which removes DNA damage from the genome overall, and TCNER, which rapidly repairs DNA lesions located specifically on the TS of active genes (for a comprehensive review of human NER, see Ref. 4 ).

A series of investigations has clearly demonstrated that the capacity of human cells to efficiently repair UV-induced CPD via NER requires the presence of a functional p53 tumor suppressor (5, 6, 7, 8, 9) . p53 apparently stimulates CPD removal through transactivation of genes that encode proteins involved in the NER process (10, 11, 12) and, in addition, may also positively or negatively regulate DNA repair by directly binding or by up-regulating proteins that directly bind to essential NER proteins (13, 14, 15) . There is major interest in the latter regard concerning the cyclin-dependent kinase inhibitor p21, which is transcriptionally up-regulated by p53 after exposure to DNA-damaging agents and was initially shown to play a key role in cellular growth arrest by preventing phosphorylation of retinoblastoma protein (16) . Indeed, p21 has been postulated to inhibit both the gap-filling step of NER and semiconservative DNA replication by binding the DNA polymerase {delta}/{epsilon} auxiliary factor PCNA that is required for each of these replicative processes (17 , 18) .

The precise role of p21 in NER remains controversial. Early studies (19 , 20) that specifically measured PCNA-dependent DNA repair synthesis in human cell-free extracts concluded that p21 has no effect on NER, although this protein was able to significantly inhibit normal DNA replication. Using a similar in vitro approach but in contrast, it was subsequently shown that p21 strongly interferes with NER gap filling, as well as with semiconservative DNA replication (21) . A more recent investigation confirmed an inhibitory role for p21 in PCNA-dependent NER gap filling in vitro and at the same time demonstrated essentially the same effect in vivo by quantifying levels of unscheduled DNA synthesis in primary human cells after introduction of purified p21 and PCNA proteins (22) . The overall issue has been further complicated by investigations in intact human tumor cells that suggest that p21, when expressed at high levels, may actually stimulate rather than inhibit the removal of UV-induced genotoxic damage (23 , 24) . Finally, using a strand-specific DNA repair assay measuring the removal of anti-CPD antibody-binding sites from chromosomal genes in vivo, it was recently shown that p21 does not influence the excision of UV-induced CPD in murine embryonic fibroblasts (25) .

In this study, we undertook to investigate whether endogenous p21 expression can modulate DNA repair specifically in the case of p53-deficient human tumor cells irradiated with UVB light. The possibility that p21 might influence the removal of UVB-induced DNA photoproducts in the absence of functional p53 is of interest, because p53 inactivation constitutes an early event in the majority of sunlight-associated human nonmelanoma skin tumors (26) . We used the highly sensitive LMPCR to directly measure at nucleotide resolution the excision rate of UVB-induced CPD from the transcriptionally active c-jun proto-oncogene in the well-characterized p53-/-p21+/+ human adenocarcinoma cell line DLD1 and in its isogenic p53-/- counterpart carrying a homozygous knockout for p21 (hereafter referred to as p21+/+ DLD1 and p21-/- DLD1, respectively). Because p21+/+ DLD1 carries only mutant p53, this strain expresses a very low basal level of p21 that is generally not up-regulated after mutagen exposure (24 , 27 , 28) . As such, p21+/+ DLD1 has been considered functionally p21-deficient in the cellular response to genotoxic damage. Nonetheless, in this study, we were able to show that low levels of endogenous p21 can significantly inhibit the capacity of p53-deficient DLD1 cells to repair UVB-induced genotoxic damage.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Cell Strains and UV Irradiation Conditions.
The p21+/+ human adenocarcinoma strain DLD1 and its isogenic p21-knockout derivative (both kindly provided by Dr. B. Vogelstein, Johns Hopkins University, Baltimore, MD) were maintained in McCoy’s 5A medium supplemented with 10% FCS plus antibiotics. The strains were initially (and periodically, thereafter) validated by ensuring that p21-specific DNA sequences could be detected by PCR only in p21+/+ DLD1. For all of the experiments in the current study, cells were grown to approximately 50–70% confluence and irradiated with UVB (290–320 nm) at room temperature after replacing the medium with 0.9% NaCl. The UVB source consisted of two fluorescent tubes (FS20T12/UVB/BP; Phillips) delivering a dose rate of 3.39 J/m2/s that was filtered through a sheet of cellulose acetate to eliminate wavelengths below 290 nm (Kodacel TA-407 clear 0.015 inch; Eastman Kodak).

LMPCR.
The LMPCR protocol has been described previously in detail (29) . Briefly, cells growing on 150-mm Petri dishes were irradiated with 450 J/m2 of UVB and allowed to repair for various times in freshly prepared culture medium. After the harvesting of cells, genomic DNA was immediately extracted and digested with T4 endonuclease V to incise the DNA at CPD sites. The resulting 5'-pyrimidine overhangs were then removed by photoreactivation using Escherichia coli photolyase, to generate ligatable ends. A gene-specific oligonucleotide was annealed downstream of the break site, and the set of genomic cleavage products was extended using cloned Pfu DNA polymerase. An asymmetric double-stranded linker was then ligated to the phosphate groups at the fragment termini, providing a common sequence on the 5'-end of all of the fragments. The longer oligonucleotide of this same linker, in conjunction with another gene-specific primer, was used in a PCR reaction to amplify the cleavage products of interest. These products were subjected to electrophoresis on 8% polyacrylamide gels alongside a Maxam and Gilbert sequencing ladder, transferred to nylon membranes, hybridized to a 32P-labeled gene-specific probe, and visualized by autoradiography. Each experimental condition was assayed in duplicate. A screening sequencing gel was run using a portion of the DNA to ensure that there was no significant variation between samples. The two samples were then pooled on a combined gel, and the resulting autoradiogram was analyzed using a Fuji BAS 1000 phosphorimager (Fuji Medical Systems, Stanford, CT). Each band represents a nucleotide position where a DNA strand break was induced by CPD cleavage, and the intensity of the band reflects the number of DNA molecules with ligatable ends terminating at that position. To assess proficiency in GNER and TCNER, relative repair rates for each strain (Table 1)Citation were determined along (a) the TS of the c-jun proto-oncogene between nucleotides +29 to +1857; and/or (b) the NTS of c-jun between nucleotides -145 to +1756. The LMPCR primer sets used for studying repair at the c-jun locus have been described previously (30) .


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  		Table 1 CPD removal in isogenic p53-deficient DLD1 colon carcinoma cell lines differing only in p21 status

 
Clonogenic Survival Assay.
p21+/+ and p21-/- DLD1 cells, at 50–70% confluence on 60-mm dishes, were irradiated with various doses of UVB light. Cells were immediately trypsinized, counted with a hemocytometer, diluted appropriately, and seeded on 100-mm dishes. After 12 days, colonies were counted after staining with 50% v/v methanol containing 0.5% w/v methylene blue.

Cellular Proliferation Assay.
Determination of cellular proliferation was achieved on UVB-exposed DLD1 cells at 50–70% confluence on 60-mm dishes by indirect BrdUrd immunofluorescence. BrdUrd (10 µM; Boehringer) was added to the culture media for 2 h preceding each time point. Cells were washed with PBS/50 mM EDTA, collected in 1 ml of PBS/EDTA, and fixed by the addition of 3 ml of ice-cold 100% ethanol. Fixed cells were centrifuged and resuspended in 1 ml of 2 N HCl containing 0.5% Triton X-100 (v/v) and incubated at room temperature for 30 min followed by centrifugation. Cells were then resuspended in PBS plus 1% BSA (w/v) and 0.5% Tween 20 (v/v) and incubated at room temperature for 1 h with 10 µl of the monoclonal IgG1 anti-BrdUrd antibody (Becton Dickinson). Cells were washed with PBS/BSA/Tween 20, incubated with 10 µl of FITC-labeled antimouse antibody (Sigma Chemical Co.) at room temperature for 30 min, centrifuged, and resuspended in PBS containing 5 mg/ml of PI (Molecular Probes). Bivariate analysis was performed using a FACScan flow cytometer (Becton Dickinson).

Apoptosis Determination.
The early apoptotic fraction was determined by annexin V/PI labeling using a kit according to the manufacturer’s specifications (R&D Systems). Briefly, cells on 60-mm dishes at 50–70% confluence were irradiated with 450 J/m2 UVB, followed by collection of adherent and floating cells at various times after treatment. Cells (25 x 104/analysis) were incubated at room temperature for 15 min in 50 µl of binding buffer containing annexin V/PI. Cells were then diluted with 150 µl of binding buffer, and bivariate analysis was performed using a FACScan flow cytometer (Becton Dickinson).


   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
LMPCR is a highly sensitive genomic sequencing method that has been adapted for measuring DNA repair rates in vivo at nucleotide resolution in chromosomal genes after exposure to genotoxic agents such as UV light (31) . We used this technique to assess the influence of endogenous p21 protein on the removal of UVB-induced CPD in human tumor cells lacking functional p53. The well-characterized p21+/+ adenocarcinoma line DLD1 and its isogenic p21-/- counterpart were used for this purpose. DLD1 bears inactivating mutations in both copies of the p53 tumor suppressor gene and has been shown to express extremely low levels of endogenous p21 that are not up-regulated after exposure to DNA-damaging agents (28) . Indeed, as demonstrated previously (27) , we were virtually unable to detect any p21 protein in untreated p21+/+ DLD1 via Western blotting (data not shown). This is consistent with studies on other p53-deficient human cell strains, which similarly display extremely low or undetectable levels of basal p21 protein expression (32, 33, 34) . Moreover, although it has been reported that p21 can, under certain circumstances, be induced by UV light independently of p53 (35, 36, 37) , nonetheless, p21+/+ DLD1 failed to manifest any detectable p21 protein accumulation up to 32 h after irradiation with 450 J/m2 of UVB, i.e., under the irradiation and growth conditions used to measure DNA repair in the current investigation (data not shown).

Fig. 1Citation and Fig. 2Citation depict representative LMPCR autoradiograms that reflect the rate of CPD removal along portions of the TS and the NTS, respectively, of the c-jun proto-oncogene in p21+/+ DLD1 and p21-/- DLD1 cells treated with 450 J/m2 UVB. In all, for each strain, 30 dipyrimidine sites located between nucleotides -145 and +1756 along the NTS and 40 dipyrimidine sites located between nucleotides +29 and +1857 along the TS were chosen randomly from these autoradiograms and from other autoradiograms spanning different portions of c-jun (data not shown). For each site, the fraction of CPDs remaining as a function of time (i.e., 0, 2, 4, 8, 12, 24, and 32 h after UVB irradiation) was quantified using a phosphorimager and displayed graphically. The times required to achieve 50% repair for all of the analyzed sites were then extrapolated (summarized in Fig. 3Citation ), pooled, and averaged for each strand. This analysis revealed a significant delay in CPD removal from both strands of c-jun in p21+/+ DLD1 relative to p21-/- DLD1, i.e., reductions of 2.1- and 2.4-fold in the average time needed to achieve 50% repair for sites along the TS and NTS, respectively (Table 1)Citation . As expected from previous DNA repair studies using LMPCR in primary human fibroblasts (31) , considerable site-to-site variation in repair capacity was observed along the c-jun gene in DLD1 tumor cells (Fig. 3)Citation . Moreover, whereas most individual sites along the NTS and TS of c-jun in p21+/+ DLD1 were repaired significantly more slowly relative to the p21-/- counterpart, some sites manifested little or essentially no difference.



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  		Fig. 1. Repair of UVB-induced CPD at nucleotide resolution along a portion of the TS of the human c-jun proto-oncogene. Repair rates at individual sites (nucleotides +318 to +392) are depicted for p21+/+ DLD1 cells (left side) and for the p21-/- DLD1 derivative (right side). The four center lanes show LMPCR of DNA treated with standard Maxam-Gilbert cleavage reactions. On either side of the sequencing ladder are eight lanes showing LMPCR of DNA isolated from UVB-irradiated cells that have undergone repair for the indicated times. The far right and far left lanes show LMPCR of unirradiated DNA followed by T4 endonuclease V/photolyase digestion. The arrows indicate dipyrimidine sites that were quantified using a Fuji BAS 1000 Phosphorimager, equipped with the Image Gauge V3.0 program. Visualization of CPDs remaining after 24 h on the TS strand in p21-/- DLD1 is precluded, because too little DNA was inadvertently loaded in this lane. However, this did not interfere with the precise determination of 50% repair times, because these times are very rapid on the TS, i.e., less than 12 h for all of the 14 sites chosen for analysis from this representative autoradiogram (Table 1)Citation .

 


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  		Fig. 2. Repair of UVB-induced CPD at individual nucleotide positions along the NTS of c-jun. Repair rates at individual sites between nucleotides +1788 to +1880 are depicted for each DLD1 derivative. The lane designations and arrow indications are the same as for Fig. 1Citation .

 


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  		Fig. 3. Summary of DNA repair rates at individual nucleotide positions along the c-jun proto-oncogene (nucleotides -145 to +1857). The times needed to achieve 50% removal of CPD along both strands of c-jun in each DLD1 derivative were extrapolated for each dipyrimidine site along the sequence, as described in the text; /, break in the sequence; {circ}, p21-/- DLD1; •, p21+/+ DLD1.

 
We emphasize that the divergence in NER rates between p21+/+ DLD1 and p21-/- DLD1 observed here does not reflect any disparity in growth rates between the two strains, i.e., resulting in possible dilution of the LMPCR signal attributable to newly replicated (nondamaged) DNA generated during post-UV incubation. Fig. 4Citation depicts the quantification of cells exposed to 450 J/m2 UVB, followed by double-staining with BrdUrd and PI and analysis by flow cytometry. The fraction of BrdUrd-positive cells actually decreased in both strains in an equivalent manner during the time period, and under the growth conditions, that repair of CPD was monitored. In addition, virtually no difference was noted in the frequency distribution of S-phase cells in the two strains over time, suggesting that each strain manifests a similar pattern of UVB-induced growth arrest.



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  		Fig. 4. Determination of cellular proliferation in UVB-irradiated p21+/+ versus p21-/- DLD1 cells. p21+/+ and p21-/- cells were plated to attain 50–70% confluence at the time of irradiation. Both strains were irradiated with 450 J/m2 of UVB, incubated for different times, and labeled with BrdUrd for 2 h preceding each time point. Incorporated BrdUrd was detected by flow cytometry using indirect immunofluorescence. A, dot plots of p21+/+ versus p21-/- DLD1 cells double-labeled with BrdUrd and PI. B, graphical representation of UVB-exposed BrdUrd-positive cells over time.

 
The above data show that very low levels of endogenous p21 can inhibit the removal of UVB-induced CPD in p53-deficient human tumor cells. This had physiological consequences consistent with modulation of NER capacity, including a decrease in clonogenic survival for p21+/+ DLD1 compared with the p21-/- counterpart. The doses of UVB required to reduce relative survival to 10% (extrapolated from Fig. 5Citation ) for p21+/+ DLD1 and p21-/- DLD1 cells were 435 J/m2 and 530 J/m2, respectively. This difference may be considered modest based on the observed disparity in DNA repair capacity between the two strains. However, we emphasize that in the current situation, CPD removal has been investigated specifically in p53-deficient cells, in which case apoptosis and not DNA repair is the primary determinant of clonogenic survival. Indeed, p53-deficient human and murine cells, although significantly compromised for NER capacity, are highly resistant to the cytotoxic effects of UV relative to p53-proficient counterparts because of loss of apoptotic capacity (9 , 26) . It follows that the relative decrease in DNA repair efficiency of approximately 2-fold observed here for p21+/+ DLD1 cells versus the p21-/- derivative (although ostensibly having important implications for genomic stability) may not be sufficient to completely overwhelm the powerful resistance to clonogenic death conferred by concomitant loss of p53-dependent apoptosis in each strain.



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  		Fig. 5. UVB-induced cytotoxicity DLD1 p21+/+ versus p21-/- DLD1 cells. The relative survival after treatment with various doses of UVB was calculated as the mean number of surviving colonies for irradiated cells relative to mock-irradiated controls. Each time point represents the average of at least three independent experiments.

 
In accord with the well-characterized prominent role for p53 in programmed cell death after exposure to DNA-damaging agents, we show that p21-/- DLD1 is highly resistant to UVB-induced apoptosis, with only 3% of the population staining positively for annexin V (indicating the early stages of apoptotic cell death) at 24, 48, or 72 h after irradiation (Fig. 6)Citation . In contrast, the p21+/+ DLD1 derivative was significantly more sensitive to apoptosis after UVB irradiation, with approximately 16% of the cell population staining positively for annexin V at each of the time points analyzed. We do not attribute this difference in apoptotic capacity to a possible direct role for p21 in UV-induced programmed cell death. Indeed, despite our demonstration that very low levels of p21 expression can significantly influence DNA repair in DLD1 cells, this minimal expression level may be insufficient to modulate any p21-dependent apoptotic response. Therefore, we suggest that p21+/+ DLD1 undergoes apoptosis more readily than the p21-/- counterpart after UVB exposure by virtue of the relative inability of the former strain to efficiently remove CPD from the TS of active genes. Indeed, it has been proposed that blockage of transcription constitutes a primary signal to initiate various cellular responses to DNA damage, including programmed cell death (38) . In support of this, it was demonstrated that after UV treatment, TCNER-deficient Cockayne syndrome cells undergo apoptosis much more readily than their DNA repair-proficient counterparts or than XP-C cells that are defective only in GNER (39 , 40) .



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  		Fig. 6. Apoptosis in UVB-irradiated p21+/+ versus p21-/- DLD1 cells. Subconfluent cells were irradiated with 450 J/m2 UVB and double-labeled with annexin V and PI at the indicated times. A, annexin V/PI profiles of p21+/+ and p21-/- DLD1 cells at 0 and 24 h after UVB irradiation. Annexin V positive cells (i.e., undergoing early apoptotic cell death) are gated in the lower right quadrant of the dot plots. B, graphical representation of annexin V positive cells at 0, 24, 48, and 72 h.

 
Notwithstanding the p53-independent inhibitory effect of p21 on DNA repair rates revealed here, previous investigations have demonstrated that p53 per se plays a critical role in NER. Indeed, as cited earlier, p53 induces various downstream effectors aside from p21 and engages in multiple protein-protein interactions that together would be expected to impact both GNER and TCNER. Although a series of studies has indicated that p53 regulates only GNER (7, 8, 9) , we have recently provided evidence that p53-deficient human cells are significantly compromised for both NER subpathways (5) . Specifically, using LMPCR, we demonstrated that p53-null Li-Fraumeni skin fibroblasts required approximately 14 and 28 h to achieve 50% repair of UVB-induced CPD along the TS versus the NTS strand of c-jun, respectively, whereas these times for p53+/+ human fibroblasts were 3 and 10 h. We show here, in general agreement with the situation for p53-null Li-Fraumeni fibroblasts, that p21+/+ DLD1 cells achieve 50% CPD removal after 12 and 23 h for the TS and NTS strand of c-jun, respectively (Table 1)Citation . Remarkably, on the other hand, the p21-/- DLD1 derivative exhibits average 50% repair times of 6 and 10 h, which approximate those manifested by the normal skin fibroblasts used in the aforementioned study (5) . Taken together, these data indicate intriguingly that ablation of p21 expression can rescue, to near normal levels, the NER defect associated with p53-deficiency in human tumor cells.

The p21-mediated effect on DNA repair in DLD1 cells is apparently specific for NER, because, to our knowledge, there is no strong evidence that human cells use other pathways (e.g., photoreactivation, base excision repair, or recombinational repair) that are used by prokaryotic and other eukaryotic organisms for the repair of CPD. Furthermore, our results shed light on the mechanism whereby low levels of p21 expression inhibit NER in a p53-deficient background. Specifically, the LMPCR method used here does not measure post-excision events, including gap filling, but rather only the preceding steps of NER, including lesion recognition/incision. This is in contrast to techniques used in previous investigations on the role of p21 in human DNA repair, which specifically measured DNA resynthesis, either in cell free extracts (19, 20, 21, 22) or, in the case of living cells, by quantifying levels of unscheduled DNA synthesis (22) . Thus, our results using LMPCR indicate that p21-mediated inhibition of DNA repair in p53-deficient DLD1 cells reflects PCNA-independent aspects of the NER process, i.e., at the level of DNA lesion recognition/incision rather than of gap filling. Significant inhibition of PCNA-dependent repair by p21 may be more likely to occur in a p53-proficient background (i.e., where p21 is strongly up-regulated after DNA damage), because in vitro and in vivo studies (22) have indicated that high molar p21:PCNA ratios would be required to interfere with gap filling.

Our data also shed some light on the potential role of DNA mismatch repair proteins in TCNER of UV-induced CPD. In this respect, we note that strain DLD1 is deficient in DNA mismatch repair by virtue of a homozygous mutation in the gene encoding the G/T mismatch-binding protein hMSH6 (41) . Furthermore, a previous investigation (42) has demonstrated that other adenocarcinoma cell strains (LoVo and HCT116) bearing inactivating mutations in different mismatch repair genes (hMSH2 and hMLH1, respectively) exhibit a virtually complete defect in TCNER of UV-induced CPD. In possible contrast, the results presented here clearly demonstrate that hMSH6-mutated DLD1 cells can remove UVB-induced CPD significantly more rapidly from the TS versus the NTS of the active c-jun locus (Table 1)Citation . However, it has also been shown that transcription-coupled repair of oxidative DNA damage (presumably via the base excision repair pathway rather than via NER) is defective in strains expressing an inactive hMSH2 protein, whereas such repair is normal in hMLH1-deficent cells (43) . Thus, taken together, the data indicate that the existence of defective TCNER of UV-induced CPD in mismatch repair-deficient human cells may depend on the particular mismatch repair gene that is inactivated.

Finally, we emphasize that the majority of human tumors in vivo, like cultured DLD1 cells, bear inactivating mutations in p53 (or otherwise express a nonfunctional p53 pathway), although still retaining a normal p21 genotype (44, 45, 46) . This situation can be readily explained on the presumption that cells lacking p53 are also functionally p21-deficient, i.e., because of their inability to up-regulate the latter protein after DNA damage, thus precluding the necessity of generating p21 mutations during tumor development. Our demonstration here that DNA repair is more efficient in p21-/- DLD1 cells, relative to their isogenic p21+/+ counterparts expressing low levels of this protein, allows speculation regarding an alternative explanation; e.g., p53 inactivation is an early initiating event in solar UVB-induced skin cell transformation and subsequently plays a critical role in tumor promotion through dysregulation of p53-dependent apoptosis (26) . The NER defect in p53-mutated skin cells presumably also contributes in a highly significant manner to the initiation and promotion of UVB-induced cutaneous tumorigenesis. Therefore, the eventual acquisition of p21 mutations in p53-deficient precancerous skin cells may not favor tumor development (and, therefore, would be rarely recovered), because such mutations might effectively restore much of the DNA repair capacity lost in these precancerous skin cells through prior p53 inactivation.


   ACKNOWLEDGMENTS
 
We thank Nathalie Bastien, Isabelle Paradis, and Anne Christine Goulet for valuable technical assistance and Dr. R. S. Lloyd for supplying T4 endonuclease V.


   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 Supported by Grants (held separately by E. A. D. and R. D.) from the National Cancer Institute of Canada (with funds from the Canadian Cancer Society) and from the Medical Research Council of Canada. E. A. D. and R. D. are research scholars of the Fonds de la Recherche en Santé du Québec. Back

2 To whom requests for reprints should be addressed, at Centre de Recherche Guy-Bernier, Hôpital Maisonneuve-Rosemont, 5415 boulevard de l’Assomption, Montréal, Québec, Canada, H1T 2M4. Phone: (514) 252-3400, extension 4665; Fax: (514) 252-3430; E-mail: drobetse{at}ere.umontreal.ca Back

3 The abbreviations used are: NER, nucleotide excision repair; CPD, cyclobutane pyrimidine dimer; GNER, global NER; TCNER, transcription-coupled NER; PCNA, proliferating cell nuclear antigen; LMPCR, ligation-mediated PCR; BrdUrd, bromodeoxyuridine; PI, propidium iodide; TS, transcribed strand; NTS, nontranscribed strand. Back

Received 8/17/00. Accepted 2/28/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Brash D. E., Rudolph J. A., Simon J. A., Lin A., McKenna G. J., Baden H. P., Halperin A. J., Ponten J. A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma. Proc. Natl. Acad. Sci. USA, 88: 10124-10128, 1991.[Abstract/Free Full Text]
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