p53 inactivation by hpv16 e6 results in increased ......spontaneous mutation rates. to determine the...

[CANCER RESEARCH 55, 4420-4424, October 1. 1

p53 Inactivation by HPV16 E6 Results in Increased Mutagenesis in Human Cells

Pamela A. Havre, Jianling Yuan, Lora Hedrick, Kathleen R. Cho, and Peter M. Glazer1

Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06520-8040 Â¡P.A. H., J. Y., P. M. GJ, and Department of Pathology,

The Johns Hopkins University School of Medicine. Baltimore. Maryland 21205 Â¡L H., K. R. C.J

ABSTRACT

To study the pathways associated with genomic instability in cancer, weexamined UV-induced and spontaneous mutagenesis in clonal cell linesexpressing human papillomavirus (HPV) proteins, either high-risk(HPV16) E6 or E7 or low-risk (HPV11) E6, in comparison to the parental

RKO cells, a colon carcinoma cell line expressing only normal p53.High-risk E6 and E7 bind and functionally inactivate tumor suppressor

proteins p53 and Rb, respectively, and both disrupt the G, arrest inresponse to DNA damage. Low-risk HPV E6 proteins bind p53 with muchlower affinity than high-risk E6 and fail to mediate p53 degradation or to

disrupt the G, checkpoint. We found that cells expressing HPV16 E6 hadreduced survival and increased mutagenesis at the hpn locus when treatedwith low doses of UV. However, this analysis was complicated by theunexpected observation of a very high background of spontaneous mutagenesis in the unirradiated cells expressing the HPV16 Â£6gene. Fluctuation analysis revealed a 5-fold elevated mutation rate in the cellsexpressing HPV16 E6. HPV11 E6 conferred a 2-fold elevation in the

mutation rate, but HPV 16 E7 had no effect. The increased spontaneousmutagenesis, therefore, appeared to be mediated by p53 inactivation andto be independent of Rb (which acts downstream of p53 in the G, arrestpathway following DNA damage). Taken together, these findings suggestthat the effect of p53 inactivation on spontaneous mutagenesis is manifested at the level of DNA repair, recombination, or coupling of transcription with one of these processes instead of by an alteration in G, arrest.

INTRODUCTION

In these studies, we compared the UV-induced and spontaneous

mutagenesis of the hprt gene in RKO cells that had been transfectedwith either a control vector (RCneo) or vectors containing HPV162 E6

(RC10.2), HPV11 Â£6(RC11.6), or HPV16 E7 (RC7.6 and RC7.14)genes (1, 2). These clones were derived from RKO cells, a colorectalcarcinoma cell line expressing only wild-type p53 (1). It has beendemonstrated that cells expressing either high-risk E6 or E7 fail toarrest in G, following DNA damage (2-6). Since binding of thehigh-risk E6 protein to p53 causes its degradation, this system hasbeen used to "knockout" p53 function. The high-risk E7 oncoprotein

also binds and inactivates its target, Rb. However, the effect oftransfection with a low risk (HPV11) E6 is more subtle, since it bindsp53 with, at best, low affinity, and does not cause its degradation (7).Low-risk (HPV11) E6 fails to abrogate the p53-mediated G, check

point (8), but it has been shown to modulate the transcriptionalregulatory function of p53 (9) and may interfere with p53 DNA-

binding activity (10).Many activities have been attributed to p53, including the media

tion of G, arrest following radiation-induced DNA damage (1). p53 is

also reported to be associated with DNA helicases (11). One of these,ERCC3/XPB, a subunit of TFIIH, is involved in both excision repair and transcription, suggesting a possible involvement of p53 in

Received 5/9/95; accepted 7/31/95.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 with18 U.S.C. Section 1734 solely to indicate this fact.

' To whom requests for reprints should be addressed, at Department of Therapeutic

Radiology, Yale University School of Medicine, Hunter Radiation Therapy, 333 CedarStreet, New Haven, CT 06520-4080.

2 The abbreviations used arc: HPV16, high-risk human papillomavirus; HPV1I, low-

risk human papillomavirus; 6-TG. 6-thioguanine; NER, nucleotide excision repair.

transcription-coupled repair (12, 13). Recently, Wang el al. (13) have

demonstrated that p53 is associated with XPB in vivo and that it canbind to the XPD and CSB proteins in vitro. They also found that p53directly modulates the helicase activity of XPB and XPD, and theyreported reduced gene-specific repair of UV-induced pyrimidinedimers in p53-deficient Li-Fraumeni syndrome cells. Smith et al. (14)have also shown diminished repair activity in extracts of p53-deficientcells, and they have reported reduced host cell reactivation of UV-

damaged plasmids in such cells.Another DNA helicase observed in association with p53 is RPA

(15), which is a single-stranded DNA-binding protein involved in

DNA repair, replication, and recombination. In particular, RPA hasrecently been implicated as a critical factor in DNA damage recognition in conjunction with the XPA and XPG proteins (16). In addition, p53 has recently been shown to be indirectly involved in balancing DNA repair and replication by its regulation of the growth-

arrest proteins, GADD45 (17) and WAF1/CIP1 (18), both of whichinteract with proliferating cell nuclear antigen, a cofactor in both DNArepair and replication in mammalian cells (19). Through its association with proteins involved in DNA repair, replication, and recombination, p53 may coordinate the interaction between these cellularactivities to maintain genomic integrity.

We report here experiments in which overexpression of HPV E6and E7 proteins was used to probe pathways associated with inducedand spontaneous mutagenesis. We find that inactivation of p53 byHPV 16 E6 leads to reduced survival and enhanced mutagenesisfollowing low doses of UV. In non-UV-treated cells, we show that E6

but not E7 expression causes an increase in the spontaneous mutationrate in cells otherwise containing normal levels of p53. Since both p53and pRB have been implicated in the G, cell cycle checkpoint pathway, we propose that the observed enhancement of spontaneousmutagenesis is a consequence of the loss of p53 function in repair.

MATERIALS AND METHODS

Cells. The parental RKO cells, a colorectal carcinoma cell line, and clonestransfected with HPV16 E6, HPV11 E6, and HPV16 E7 were describedpreviously (1-3). Cells were maintained in McCoy's 5A medium/10% PCS

(GIBCO-BRL, Bethesda, MD). Media for transfected cells was supplementedwith 0.5 mg/ml G418 (GIBCO-BRL).

UV-induced Mutagenesis and Cytotoxicity. RKO cells, either transfected

with the pCMVneo vector alone (RCneo) or with the vector containing thehigh-risk (HPV16) E6 gene (RC10.2), were seeded at 1 X 10" cells/100-mm

dish. Later the same day, the media was removed, cells were irradiated withUV from 0 to 25 J/m~, using a 254 nm germicidal lamp as measured by an

IL 1400 radiometer from International Light (Newburyport, MA), and the oldmedia was replaced. To measure cytotoxicity, the cells were then detached bytrypsinization, diluted with fresh medium, and distributed to dishes for determination of colony formation. Induced mutagenesis was determined in separate cultures by the addition of 40 JU.M6-TG (20) at a cell density of 1 X IO6/

dish 4 days after irradiation. Approximately 10 days following irradiation, cellswere stained with 0.25% cresyl violet, and colonies containing 30 or more cellswere counted either as surviving clones (in the cytotoxicity assay) or asmutant clones (in the mutagenesis assay following 6-TG exposure).

Spontaneous Mutation Frequency. The mutation frequency was determined for RCneo and RC10.2 by treating 1 x 10'' cells in a preexisting culture

(not cloned) with 6-TG as described above, and 6-TG-resistant colonies were

counted 10 days later.

4420

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MUTAGENKSIS IN HPV E6-TRANSH-CTF.D CELLS

Spontaneous Mutation Rates. To determine the spontaneous mutationrate and to obtain clones for DNA isolation, each cell type was cloned by

limiting dilution and then expanded. For the cell lines RCneo (21 clones),RC10.2 (15 clones), RC11.6 (10 clones), and either RC7.6 or RC7.14 (13clones), cells were incubated with 6-TG as described above and stained 10

days later. The spontaneous mutation rate was calculated by fluctuation analysis using the method of the mean (21, 22). Unstained duplicate plates wereused for isolation of 6-TG-resistant clones to be expanded for the isolation of

DNA.Analysis of Mutant Clones for Exons 1, 3, and 9 of the hprt Gene Using

Multiplex PCR Amplification. Forty 6-TG-resistant clones were isolated andexpanded to 10" cells. DNA was isolated and amplified using primers and

conditions for amplification as described (23), except that exons 3 and 9 wereamplified together and exon 1 was kept separate. PCR products were analyzedon 1.5% agarose gels using a l(K)-bp ladder to determine the size of the

product, and the expected bands corresponding to the exons were scored aspresent or absent.

RESULTS

Mutagenesis Frequency and Cell Survival following UV Irradiation. A comparison of the survival curves for the RCneo controlcells and the RC10.2 cells expressing HPV16 E6 is presented in Fig.LA. At low to moderate doses, there appears to be a differencebetween the cell lines, with RCneo cells showing greater survival.Because the overall curves converge at high doses, an exponentialcurve fit eliminated the difference between the cell lines (data notshown). However, the difference at moderate doses was reproduced inthree separate experiments. These results provide evidence that theRC10.2 cells may have a subtle repair defect that is manifest at lowlevels of UV-induced damage, leading to reduced survival relative to

the RCneo cells at the lower doses. The curve for the RC10.2 cellsmay also have a reduced shoulder in the low dose range, although thisregion of the survival curve was not examined in detail. Nonetheless,this could also be a sign of a diminished repair capacity. However, athigher doses of 15 J/m2 or more, the survival curves come together. At

these high UV doses, the differences in the repair capacities of the twocell lines may be obscured by the excessive damage, leading to similarlow levels of survival. Also, since p53 has been implicated in theinduction of apoptosis (24), the functional Â¡nactivationof p53 in theRC10.2 cells may give them a relative growth advantage underconditions that might otherwise induce apoptosis, such as high levelsof UV-induced DNA damage. Hence, the RC10.2 cells, even with

reduced repair capacity, might show relatively better survival in thisdose range because of a resistance to apoptosis. However, we have notbeen able to detect the induction of apoptosis in the RKO-derived cell

lines under the conditions of this experiment (data not shown), and inother work, we have found that RKO cells are not prone to apoptosis.

Using the hprt locus to assess mutation frequency, UV-induced

mutagenesis in the RC10.2 cells and RCneo control cells was analyzed (Fig. IÃŸ).RC10.2 cells, which express the high-risk E6 onco-protein, exhibited higher frequencies of UV-induced mutations than

the control RCneo cells expressing functional p53, especially at lowdoses of irradiation. This is consistent with the suggestion, based onthe survival curve data, that there is a difference in repair capacitiesbetween these cell lines. It is also consistent with other studiesshowing a direct effect of p53 on DNA repair (12-14, 17). At the

higher UV doses, the mutation frequency curves converge, suggestinga saturation of the repair process. However, the greatest difference inmutagenesis between the two cell lines was seen in the absence ofirradiation. This unusually high frequency of mutagenesis in theunirradiated RC10.2 cells prompted us to further investigate spontaneous mutagenesis in these cell lines.

.001

dose (J/m2)

B100000

(O

UV Dose (J/m2)

Fig. 1. Cell survival and mutagenesis following UV irradiation. RCneo (control) andRC10.2 (high-risk E6) were irradiated with 0-25 J/nr of UV radiation. A. clonogenicsurvival. B. UV-induced mutation frequency in the hpr! gene. The number of mutants wascorrected for survival at each UV dose, and the mutation frequency was then calculatedon a per survivor basis.

Determination of Spontaneous Mutation Frequencies for RCneo and RC10.2 Cells. The mutation frequency is calculated as thepercentage of mutations occurring in preexisting cultures. In contrast,the mutation rate is determined by starting with a small number ofcells with no preexisting mutations and measuring the occurrence ofmutations during growth of the cells in culture (22). In an experimentto measure spontaneous mutation frequencies, replicate cultures ofRCneo and RC10.2 cells were seeded at a density of 1 X IO6

cells/l()()-mm dish, and following attachment of the cells, 6-TG was

added to a final concentration of 40 JU.M.The resistant colonies werecounted 10 days later and expressed as the average number of resistantcolonies (mutants) per 10'' cells (Table 1). The mutation frequency for

RC10.2 was 73-fold higher than for RCneo. The large difference

between these two cell types is attributed to the presence of HPV 16E6 and the consequent inactivation of p53 in RC10.2.

4421



MUTAGENESIS IN HPV E6-TRANSFECTED CELLS

Table 1 Frequency of spontaneous mutations in RCneo and RCÃŒ0.2(high-risk E6) cells

Cell typeMutation frequency (X10 )"

RCneo (control)RC10.2 (high-risk E6)

2.5182.7

" The mutation frequency was obtained by counting 6-TG-resistant colonies 10-14days after addition of the purine analogue to 1 X 10 cells taken from a pre-existing

culture.

Table 2 Spontaneous mutation rates for RKO cells and E6 and El transfeclants

CelllinesRCneoRC10.2RC11.6RC7.6

and 7.14TransfectedHPV

geneNoneHigh-risk

E6Low-riskE6E7No.

ofreplicatecultures21151013Mutationrate"(X10~6)3.1815.367.083.56

" To determine spontaneous mutation rates (mutations per IO6 cells/generation), the

indicated cells were cloned by limiting dilution. Replicate cultures (the numbers of whichare indicated) from the individual clones from each cell line were expanded to 1 X 10cells and were exposed to 6-TG. Surviving colonies (mutants) were counted 10 days later,and the spontaneous mutation rate was calculated by the method of the mean (21, 22).

Spontaneous Mutation Rates in Parental RKO cells and E6 andE7 Transfectants. Because the above results reflect the previousaccumulation of mutations during passage of the cells in culture, wedecided to measure the spontaneous mutation rates. In addition toRC10.2 which expresses the high-risk E6 oncoprotein, two other

transfectants were used in the comparison. RC11.6 cells express thelow-risk (HPV11) E6 protein, whereas RC7.6 and RC7.14 express thehigh-risk (HPV 16) E7 gene. These latter cells, in which E7 oncopro

tein expression causes pRb inactivation, fail to arrest in G, following

DNA damage (2, 3).In this experiment, the cell lines were cloned by limiting dilution.

Multiple clones for each cell line were independently expanded andthen seeded at a cell density of 1 X IO6 cells/dish in the presence of

6-TG. Surviving clones (mutants) were counted after 10 days as

described above.Using the method of the mean (21, 22), quantitation of the accu

mulated mutants arising in the replicate cultures for each cell lineallowed calculation of the mutation rates (Table 2). Cells containingthe high risk E7 oncoprotein (RC7.6 and RC7.14) behaved like thecontrol RKO cells containing the pCMVneo vector only with amutation rate of 3.6 versus 3.2 per IO6 cells/generation (Table 2). In

contrast, cells containing either the low-risk E6 (RC11.6) or thehigh-risk E6 (RC10.2) had higher mutation rates of 7.1 and 15.4 perIO6 cells/generation, respectively. Therefore, p53 but not Rb appears

to be responsible for maintaining a normal level of mutagenesis inthese cells. This observation suggests that G! arrest is not solelyresponsible for protection of the genome from the accumulation of

mutations.Analysis of Mutants from Parental RKO Cells and E6 and E7

Transfectants. Exons 1, 3, and 9 of the hprt gene were analyzed byPCR amplification of DNA isolated from 40 different 6-TG-resistant

clones. Exons 3 and 9 were amplified in one reaction and exon 1 in aseparate reaction. The products were analyzed by agarose gel elec-

trophoresis to determine the presence or absence of the appropriateband for each exon. In the case of RC10.2, in which p53 is inactivatedby degradation, deletions of at least one exon were detected in 8 of 14mutants (Table 3). For the other three lines, only 2 deletions of 26were seen. From this analysis, the trend is that the inactivation of p53leads to an increased spontaneous mutation rate, generating both pointmutations and deletions and/or rearrangements in the hprt gene.

DISCUSSION

In this work, we have compared the UV-induced response of

control cells with that of cells in which p53 or pRb function wasdisrupted by expression of HPV E6 or E7 oncoproteins, respectively.We found that the cells expressing HPV16 E6 showed a reducedsurvival and an elevated mutation frequency in response to low dosesof UV irradiation. A similar difference in survival after UV irradiationbetween RKO cells and RKO cells transfected with HPV E6 wasreported by Smith et al. (14). There is a discrepancy between theirresults and ours in that they did not see a convergence of the survivalcurves at doses above 15 J/m2. However, they tested only one dose

point above this level, and we tested only two, and so this point hasnot been extensively analyzed. Nonetheless, our results confirm theirobservation that E6-mediated p53 inactivation influences survival at

moderate UV doses, suggesting a role of p53 in the repair of UVdamage. A previous study with ionizing radiation did not show acorrelation of p53 status with clonogenic cell survival (25). Thepotential reasons for this difference are discussed in detail by Smith etal. (14), and they may reflect the differences between X-ray andUV-induced DNA damage as well as differences in the corresponding

cellular repair pathways.In this regard, Fan et al. (26) found that disruption of p53 in MCF-7

breast carcinoma cells sensitized the cells to cisplatinum but not toionizing radiation or to certain other types of DNA-damaging agents.Like cisplatinum-induced adducts (but unlike damage from ionizingradiation), UV-induced lesions are repaired primarily through the

NER pathway. Hence, in RKO colorectal carcinoma cells and inMCF-7 breast carcinoma cells, p53 status may play an important role

in the response to DNA damage that is repaired by NER. In other celltypes, however, in which p53-mediated apoptosis may be more prom

inent, such as lymphoid cells, inactivation of p53 appears to conferincreased resistance to most types of DNA damage (27-29). Hence,the relative importance of p53-stimulated repair versus p53-dependent

apoptosis in response to DNA damage may depend on the cell typeand on the nature of the damage.

With regard to mutagenesis, our results show that p53 inactivationis associated with a small increase in UV mutation frequency atmoderate UV doses, again consistent with the role of p53 in DNArepair. Smith et al. (14) did not examine mutagenesis, but they didfind that cells expressing HPV16 E6 showed reduced host cell reactivation of a UV-damaged plasmid. Similarly, Fan et al. (26) alsofound decreased reactivation of a cisplatinum-damaged plasmid upon

p53 inactivation. These complementary experiments demonstrate thatp53 is an important component of the NER pathway that removes UVdamage. The results fit well with emerging data that p53 directly bindsto or interacts with factors in this pathway, such as XPB, XPD, RPA,and proliferating cell nuclear antigen (13, 15, 17).

In the course of our investigation of UV-induced mutagenesis,

however, we observed an abnormally high frequency of spontaneousmutations in RKO cells expressing the high-risk E6 gene (RC10.2).

Table 3 Increased frequency of hprt gene deletions and/or rearrangements in RKOcells transfected with HPVI6 E6 or E7 and HPVll E6

CelllineRCneoRC10.2RC11.6RC7.6No.of deletions orrearrangements"18(I1Total clonesanalyzed171454

" 6-TG-resistant colonies arising during clonal expansion of the indicated cell lines

were isolated. Genomic DNA from the mutant colonies was analyzed by multiplex PCRamplification to evaluate the hprt locus at exons 1, 3, and 9. Deletions and/or rearrangements were detected by the absence of the appropriate amplified DNA fragments in thePCR reactions as visualized by agarose gel electrophoresis.

4422



MUTAGENESIS IN HPV E6-TRANSFECTED CELLS

Measurement of the rate of spontaneous mutagenesis revealed a 5-folddifference between the RKO control cells and RC10.2 and a 2-folddifference between RKO and RC11.6, which expresses the low-riskE6 gene. Expression of the high-risk E7 gene did not alter the

spontaneous mutation rate. Therefore, our data support a role for p53but not Rb or Rb-related proteins p 107 or p 130 in the maintenance of

a low mutation rate. Moreover, our data are consistent with previousresults suggesting that low-risk E6 proteins can, to some extent,disrupt p53 function, albeit to a lesser extent than the high-risk HPV

E6 oncoproteins (9, 10). Because both Rb and p53 play a role in cellcycle control, we interpret these results as reflecting the role of p53 ininfluencing DNA repair and replication apart from its effect on theG,-S transition, again in agreement with reports of direct interactions

of p53 with repair proteins (13).Using multiplex PCR analysis of genomic DNA, we found that

57% of the mutations arising spontaneously in the cells expressingHPV16 E6 were deletions of at least one exon, whereas the othersappeared to be point mutations. Only 7.7% of the mutations in theother cell lines were found to be deletions. One explanation for this isthat aberrant or diminished repair of endogenous DNA damage maylead not only to point mutations but also to deletion-prone interme

diates, such as strand breaks or gaps, representing discontinuities thatmay arise during stalled replication of the damaged DNA template.Consistent with this hypothesis is the observation that inactivation ofp53 via E6 expression causes an elevation of the spontaneous recombination frequency between tandemly repeated genes in diploid human fibroblasts.3

In contrast to our results, other studies of the influence of E6 and E7oncoproteins on the maintenance of genomic integrity have detectedeffects resulting from the inactivation of both p53 and Rb (30). Thishas led to the proposal that the joint role of p53 and Rb in G, arrestis central to the preservation of genome stability. However, the endpoints of these other studies were different, focusing on gross chromosomal alterations such as gene amplification, chromosome rearrangements, and aneuploidy rather than gene-specific mutagenesis.

Nonetheless, the differences in the results suggest that several mechanisms may be important in maintaining the integrity of the genome,with respect to both gene-specific mutations and to chromosome

structure and organization.The type of analysis used in this work to measure the mutator effect

of E6 expression and consequent p53 inactivation in human cells hasbeen used to quantitate the mutator phenotype in other human cancercells. Bhattacharyya et al. (31) observed an abnormally high mutationrate for three human colorectal carcinoma cell lines deficient in DNAmismatch repair. All three of these cell lines displayed an elevatedmutation rate at the hprt locus, ranging from 1.5 to 2.6 X 10 "5 per

cell/generation. These rates are comparable to the rate observed forthe RKO cells transfected with HPV16 E6 (RC10.2), which was1.5 X 10~5. The mutation frequency was also elevated at the hprt

locus in the mismatch repair-deficient lines, ranging from 6 to40 X IO"4. For RC10.2, we observed a frequency of 1.8 X 10~4compared to the frequency of 2.5 X 10"'' in the control RKO cells.

Hence, in the hprt mutation assay, the effect of p53 inactivation is notas large but is in the same range as the effect of a defect in mismatchrepair.

Both the high- and low-risk papillomaviruses can cause prolifera-tive epithelial lesions, but only the high-risk types are associated with

the development of invasive cervical cancers. The inability to obtaina fully transformed, malignant phenotype with low-risk HPVs sug

gests that other events besides cell proliferation must occur to allow

development of the cancerous state. Since these events could bemutations, which would then accumulate over time (32), a cell phenotype that exhibits a substantially higher than normal spontaneousmutation rate would be more prone to transformation to a fullydeveloped cancer cell.

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1995;55:4420-4424. Cancer Res Pamela A. Havre, Jianling Yuan, Lora Hedrick, et al. in Human Cellsp53 Inactivation by HPV16 E6 Results in Increased Mutagenesis

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