induced cyclobutane pyrimidine dimers (CPDs) from a. 16 Kb fragment of the p53 gene. In NHOK the NER activity was initiated in both DNA strands immediately,.
Oncogene (1999) 18, 6997 ± 7001 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc
Impaired nucleotide excision repair in UV-irradiated human oral keratinocytes immortalized with type 16 human papillomavirus genome Osvaldo Rey1,2, Sora Lee1 and No-Hee Park*,1,2 1
UCLA School of Dentistry, Los Angeles, California, CA 90095-1668, USA; 2Dental Research Institute, Los Angeles, California, CA 90095-1668, USA
We previously reported that `high risk' human papillomaviruses (HPV) induce genetic instability in human oral keratinocytes. To understand the mechanisms of HPVinduced genetic instability, we determined the nucleotide excision repair (NER) capacity of normal (NHOK) and human papillomavirus type-16 immortalized oral keratinocytes (HOK-16B) by strand-speci®c removal of UVinduced cyclobutane pyrimidine dimers (CPDs) from a 16 Kb fragment of the p53 gene. In NHOK the NER activity was initiated in both DNA strands immediately, although the process in the non-transcribed strand was notably slower than that of the transcribed strand. In HOK-16B cells the initiation of CPDs removal was delayed for at least 8 h in both DNA strands, and the process was signi®cantly slower than that in NHOK. UV-irradiation enhanced the p53 protein level more than 30-fold in NHOK, but it did not signi®cantly alter the protein level in the HOK-16B cells. UV-irradiation also increased the p21WAF1/CIP1 protein level only in NHOK. These data indicate that `high risk' HPV induces genetic instability by impairing NER capacity of cells. Impaired NER activity of HOK-16B cells may be implicated with their inability to enhance active p53 when challenged by genotoxic stress. Keywords: HPV; p53; oral cancer; DNA repair
Introduction Disruption of DNA repair may eventually result in the loss of genetic integrity, a hallmark of neoplastic cells. We previously reported that `high risk' human papillomaviruses (HPV) induced genetic instability in human oral keratinocytes (Shin et al., 1996). Although the detailed mechanisms of HPV-induced genetic instability remain to be investigated, both E6 and E7 oncoproteins of the `high risk' HPV may be responsible for the instability: They are mutagenic and disrupt the DNA replication in human oral keratinocytes (Liu et al., 1997). These oncoproteins also negate the cell cycle control by inactivating tumor suppressor p53 and pRB proteins when cells are challenged by genotoxic stress (Liu et al., 1997): The E6 oncoprotein binds and enhances the degradation of p53 via a ubiquitinmediated mechanism (Band et al., 1993; Foster et al., 1994; Hubbert et al., 1992; Schener et al., 1990). The
*Correspondence: N-H Park Received 30 April 1999; revised 16 August 1999; accepted 23 August 1999
HPV E7 oncoprotein inactivates the pRB protein (Tommasino et al., 1993). The tumor suppressor p53 protein is involved with nucleotide excision repair (NER) (Bakalkin et al., 1994; Coverley et al., 1992; Dutta et al., 1993; Wang et al., 1994, 1995), the main repair mechanism of UV-induced DNA damage (Aboussekhra and Wood, 1994; Sancar, 1994; Sancar and Tang, 1993). This NER pathway is divided into transcription-coupled repair (TCR), which is restricted to the transcribed strand of transcriptionally active genes, and general genome repair, which acts on DNA lesions within the entire genome. After exposure to DNA damaging agents, the level of cellular p53 protein increases rapidly by a post-translational enhancement mechanism (Fritsche et al., 1993). However, there is evidence to suggest that p53 may not be necessary in the NER process of all cell types (Ishizaki et al., 1994). To investigate the mechanism of HPV-induced genetic instability in human oral keratinocytes, we compared the NER activities of NHOK and HPVimmortalized human oral keratinocyte cell line after UV-irradiation. We found that the NER in both transcribed and non-transcribed strands of the p53 gene was signi®cantly delayed and lower in HOK-16B cells compared with NHOK. UV-irradiation notably increased the levels of p53 and p21WAF1/CIP1 in NHOK, but it failed to alter the levels of these proteins in the HOK-16B cells. These data indicate that `high risk' HPV induces genetic instability by impairing NER capacity of cells. Impaired NER activity of HOK-16B cells may be implicated with their inability to enhance active p53 when challenged by genotoxic stress. Results Analysis of total RNA isolated from HOK-16B cells showed that transcripts with the potential to encode the E6 and E7 oncoproteins were present in these cells (Figure 1). UV-irradiation did not signi®cantly alter the level of this viral transcript immediately or within 3 h after UV-irradiation (Figure 1). However, we were unable to detect the E6 oncoprotein in these cells by radiolabeling and immunoprecipitation (Figure 2) or by Western blot analysis (data not shown). In contrast, the viral E7 oncoprotein was readily detected under the same conditions (Figure 2). The NER activity of HOK-16B cells after UVirradiation was compared with that of NHOK under the same condition. The strand-speci®c removal of UVinduced CPDs from a 16 Kb EcoRI fragment of the p53 gene was analysed using single-stranded labeled riboprobes. Within 8 h post-UV exposure, approxi-
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Figure 1 E6/E7 RNA expression in HOK-16B cells. Total RNA (15 mg/lane) isolated from NHOK or HOB-16B cells after UVirradiation was resolved in a 1% agarose-formaldehyde gel and transferred to nylon membranes. The E6/E7 transcripts on the membranes were hybridized with a32P-labeled probes synthesized as described in Materials and methods. Signals were detected by exposing a phosphorimager screen to the probed membranes before quanti®cation. The obtained values were normalized by comparison with the GPDH transcript content. A.U., arbitrary units; hpUV: hours post-UV irradiation
Figure 2 E6 and E7 protein expression in HOK-16B cells. Total protein extracts obtained from radiolabeled NHOK and HOK16B cells were immunoprecipitated with antibodies against HPV16 E6 or E7 proteins and electrophoresed on a SDS ± 12% PAGE. Detection was performed with a phosphorimager
mately 60% of the transcribed and 20% of the nontranscribed DNA strands were repaired in NHOK, whereas less than 5% of either DNA strands was repaired in HOK-16B cells (Figure 3). By 24 h after UV-irradiation, 100 and 60% of the transcribed and non-transcribed strands, respectively, were repaired in NHOK cells compared with only 50 and 38% of these strands in the HOK-16B cells (Figure 3). When compared the level of p53 mRNA in NHOK and HOK-16B cells, the latter contained three times as much p53 mRNA as did NHOK cells. These levels were not signi®cantly aected by UV-irradiation (Figure 4a). The p53 protein level in HOK-16B cells was similar to that in NHOK prior to UV-irradiation; however, its level was not notably changed in HOK16B cells compared with a 30-fold increase in NHOK after UV irradiation (Figure 4b). To determine whether the failure of HOK-16B cells to increase the level of
Figure 3 NER activity in HOK-16B cells. Strand-speci®c removal of UV-induced cyclobutane pyrimidine dimers was analysed on a 16 kb EcoRI fragment of the active p53 gene using speci®c single-stranded a32P-labeled riboprobes. DNA was extracted from NHOK and HOK-16B at dierent hours after UV-irradiation (hpUV), digested with EcoRI and treated (+) or mock-treated (7) with T4 endonuclease V (T4V). DNA was electrophoresed on denaturing agarose gels, transferred to membranes and hybridized with strand-speci®c labeled riboprobes. Signals in the membranes were detected (a) and quanti®ed (b) using a Storm 840 phosphorimager. Removal of CPDs was calculated by comparing the amount of radioactivity in the T4V treated versus the mock-treated fragments and normalized by comparison with a loaded control plasmid. Results represent the average of three independent experiments. & DNA transcribed strand; * DNA non-transcribed strand. Bars: standard deviation
p53 protein was due to its enhanced degradation induced by HPV-16 E6, we examined the eect of a speci®c inhibitor of the proteasome, lactacystin, on p53 degradation in HOK-16B cells. As shown in Figure 5, exposure to lactacystin did not increase the p53 level in HOK-16B cells, whereas it did increase the p53 level in NHOK. There was a similar eect on the expression of p21WAF1/CIP1 protein, one of the gene products whose transcription is partly regulated by wt p53 (el-Deiry, 1998; el-Deiry et al., 1994) (data not shown). Discussion In HOK-16B cells, we did not detect the HPV-16 E6 oncoprotein, although the cell line contains approximately 25 copies of the integrated HPV-16 genome per cell (Park et al., 1991) and expresses transcripts with the potential to encode both the E6 and E7 oncoproteins. Failure to detect E6 was not due to the employed antibody since E6 was detected by radioimmunoprecipitation using the same antibody in a transient expression system (data not shown). Also, the E6 half-life, approximately 4 h, is long enough to ensure its detectability. Several possible explanations for the indetectability of E6 are: E6 expression occurred only during the early stages after integration of the viral DNA into the cellular chromosomes, E6 is synthesized in undetectable amounts or its mRNA is translationally blocked. Presence of `high risk' HPV E6 protein from several cervical cancer cell lines was demonstrated (Androphy et al., 1987), indicating that
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Figure 5 Eect of the proteasome inhibitor lactacystin on the degradation of p53 in HOK-16B cells. Total protein extracts obtained from NHOK and HOK-16B untreated or treated during 2 h with lactacystin (5 mM) were electrophoresed on SDS ± 10% PAGE and transferred to membranes. Membranes were stained using murine monoclonal antibodies against p53 and alkaline phosphatase conjugated second antibody plus a chemi¯uorescent substrate. Detection was performed with a phosphorimager. Cells incubated with DMSO were also included as controls
Figure 4 p53 expression in HOK-16B. (a) Total RNA (15 mg/ lane) isolated from NHOK and HOB-16B after UV-irradiation was resolved in a 1% agarose-formaldehyde gel and transferred to nylon membranes. The p53 transcript on the membrane was hybridized with a a32P-labeled probe synthesized as described in Materials and methods. Signals were detected by exposing a phosphorimager screen to the probed membranes before quanti®cation. The obtained values were normalized by comparison with the GPDH transcripts. (b) Total protein extracts from NHOK and HOK-16B obtained after UV-irradiation were electrophoresed on SDS ± 10% PAGE and transferred to membranes. Membranes were stained using murine monoclonal antibodies against p53, p21WAF1/CIP1 or b-tubulin and alkaline phosphatase conjugated second antibody plus a chemi¯uorescent substrate. Detection and quanti®cation were performed with a phosphorimager. The obtained values were normalized by comparison with the b-tubulin content. A.U., arbitrary units; hpUV, hours after UV-irradiation, NI, non UV irradiated cells
the protein is detectable from cells harboring `high risk' HPV genome. Moreover, the number of HPV genomes integrated into some of these tumor-derived cell lines is very high. For example, CaSki cells contains 600 copies of the HPV-16 genome compared to 25 copies in HOK-16B. Although we were not able to detect E6 in the HOK-16B cells, we noticed that an UV-induced increase of p53 was not seen in these cells. `High
risk' HPV E6 enhances the ubiquitin-mediated degradation of p53 and, in doing so, prevents the accumulation of p53 in cells when exposed to genotoxic agents (Band et al., 1993; Foster et al., 1994; Hubbert et al., 1992; Schener et al., 1990). We thus examined the eect of lactacystin, an inhibitor of the 20S proteasome involved in the ubiquitin-mediated degradation of p53 to determine if E6 is involved in the degradation of p53 in HOK-16B cells. Lactacystin notably enhanced the p53 level in NHOK, but did not increase the protein level in HOK-16B cells. This observation suggests that failure of UV-irradiation to induce an increase of p53 in HOK-16B cells may not be associated with HPV-16 E6. The mode of the failure to enhance p53 in HOK-16B cells after UVexposure remains to be investigated. It has been suggested that p53 is involved in DNA repair (Bakalkin et al., 1994; Coverley et al., 1992; Dutta et al., 1993; Wang et al., 1994, 1995). However other studies indicated that this is not so. For instance, there is no dierence in the NER activity of normal mouse ®broblasts and ®broblasts derived from p53 knockout mice (Ishizaki et al, 1994). On the contrary, the removal of CPDs from the non-transcribed DNA strand is aected in primary human ®broblasts expressing HPV-16 E6 protein (Ford et al., 1998) or in ®broblasts from Li-Fraumeni patients expressing mutant p53 (Ford and Hanawalt, 1995). Therefore it appears that the defective NER activity in HOK-16B appears to be caused by a mechanism not involving HPV E6. In HOK-16B we detected more p53 encoding transcripts and similar level of p53 protein compared with NHOK before UV-irradiation. In contrast, however, UV-irradiation increased the level of p53 protein in NHOK more than 30-fold, whereas it did not signi®cantly alter the protein level in the HOK-16B cells. A similar eect was detected for the p21WAF1/CIP1 protein whose expression is controlled in part by p53. We did not detect any mutation in the highly conserved region of p53 in HOK-16B (codons 117 ± 309) that could aect its half-life (Li et al., 1992), although we cannot rule out the existence of other mutations outside this region which could have a negative eect on its translation or stability. Thus, after UV exposure the HOK-16B cell appears to have a defect in the accumulation of p53 protein that is not dependent upon E6.
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It has been reported that HPV-16 E7 by itself can also alter genomic integrity and be mutagenic (Liu et al., 1997; White et al., 1994). Our present results suggest that the HPV-16 E7 protein can have a broader eect than previously thought possibly by a mechanism that impairs NER DNA repair and p53 translation. Whether E7 does so by aecting cellular functions downstream of E6 or by another pathway remains to be determined. Materials and methods Cell cultures Primary normal human oral keratinocytes (NHOK) and HOK-16B cells were cultured as described previously (Park et al., 1991) in keratinocytes growth medium (KGM) supplemented with reagents supplied in the bullet kit (Clonetics Corp., San Diego, CA, USA). Northern analysis Total RNA from NHOK and HOK-16B was extracted and analysed as previously described (Rey et al., 1999). The signals were quanti®ed with Image-Quant software, version 1.2 (Molecular Dynamics, Sunnyvale, CA, USA) and normalized by comparison with GAPDH transcripts. Radiolabeling and immunoprecipitation Culture of NHOK and HOK-16B cells (2610 ) incubated for 30 min in cysteine-free Dulbecco's minimal essential medium (DMEM) minus serum were pulse-labeled with 100 mCi/ml of [35S]-cysteine (ICN Biomedicals, Inc., Irvine, CA, USA) for 2.5 h. After labeling, the cells were lysed by incubation for 30 min at 48C in 1 ml of lysis solution (20 mM Tris-HCl, pH 7.6; 150 mM NaCl; 1% Triton-X100; 0.5% sodium deoxycholate; and a protease inhibitor cocktail [Boehringer Mannheim, GmbH, Germany]). Cell lysates were centrifuged at 8000 g for 10 min at 48C, and proteins were immunoprecipitated from the supernatant at 48C. The immune complexes, collected with a mixture of Protein A Sepharose (Pharmacia Biotech AB, Uppsala, Sweden) and Protein G Plus-agarose (Calbiochem-Novabiochem Corp., La Jolla, CA, USA) were washed twice with ice-cold washing solution (50 mM Tris-HCl, pH 7.6; 500 mM NaCl; 1% Triton-X100; 0.5% sodium deoxycholate; 1% BSA; and protease inhibitor cocktail) and once with the lysis solution. Protein were extracted for 10 min at 958C in 26 sodium dodecyl sulfate (SDS)-sample buer (200 mM Tris-HCl, pH 7.0; 6% SDS; 2 mM EDTA; 4% 2-mercaptoethanol; and 10% glycerol) and electrophoretically separated in 12% SDS-polyacrylamide gels. Signals were detected using a Storm 840 phosphorimager (Molecular Dynamics). 6
Probes Strand-speci®c riboprobes for the p53 EcoRI fragment detection were prepared by PCR ampli®cation of a human genomic DNA fragment between nucleotides 1750 ± 2138 of exon II of the p53 gene. This fragment of 388 nucleotides was ligated into the pGEM-T plasmid (Promega, Corp., Madison, WI, USA) and sequenced to con®rm the absence of mutations. Strand-speci®c a32P-labeled riboprobes were generated using the T7 or SP6 transcription promoters in the pGEM-T/p53 vector as previously described (Rey and Nayak, 1992). a32P-labeled DNA probes for p53 transcript detection were synthesized using a template corresponding to the complete human wild type p53 open reading frame (ORF) and the Prime-It RmT system (Stratagene, La Jolla, CA, USA). a32P-
labeled DNA probes for E6/E7 transcript detection were synthesized using a cDNA fragment encompassing nucleotides 83 ± 538 of the E6/E7 transcript (HPV-16) and the Prime-It RmT system (Stratagene, La Jolla, CA, USA). DNA repair assay Strand-speci®c removal of UV-induced cyclobutane pyramidine dimers (CPDs) was analysed from a 16 kb EcoRI fragment of the active p53 gene using single-stranded labeled riboprobes speci®c for p53 (Bohr et al., 1985; van der Horst et al., 1997). Cells cultured to con¯uence were irradiated with 2.5 J/m2 (254 nm) and genomic DNA was extracted at 0, 8 and 24 h post UV-irradiation using a QIAamp tissue kit (Quiagen Inc., Valencia, CA, USA). The extracted DNA digested with EcoRI and treated or mock-treated with T4 endonuclease V (T4V) (Epicentre Technologies, Madison, WI, USA), then electrophoresed on denaturing agarose gels and transferred to Hybond-N membranes (Amersham Life Sciences, Arlington Heights, IL, USA). After hybridization with strand-speci®c riboprobes, signals in the membranes were detected using a Storm 840 phosphorimager and quanti®ed with Image-Quant software, version 1.2 (Molecular Dynamics). Repair of CPDs was calculated by comparing the amount of radioactivity in the T4V treated versus the mock-treated fragments and normalized with a loaded control plasmid. Western analysis Total protein extracts were obtained as previously described (Rey and Nayak, 1992) from NHOK and HOK-16B cells at dierent times after UV-irradiation at 2.5 J/m2 (254 nm). Protein samples were electrophoresed on 10% SDS polyacrylamide gels and transferred to Immuno-Blot PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA). Standard procedures using alkaline phosphatase conjugated second antibody, a chemi¯uorescent substrate (Vistra Systems, Amersham Life Sciences) and a Storm 840 phosphorimager (Molecular Dynamics) were employed to detect the transferred proteins. The obtained signals were quanti®ed with Image-Quant software, version 1.2 (Molecular Dynamics) and normalized against b-tubulin content. Antibodies The following antibodies were used in this study: Ab-2, a murine monoclonal antibody speci®c for p53 (CalbiochemNovabiochem Corp.); Ab-1, a murine monoclonal antibody speci®c for p21CIP1/WAF1 (Calbiochem-Novabiochem Corp.); a murine monoclonal antibody speci®c for b-tubulin (Amersham Life Sciences); C-19, a goat polyclonal antibody speci®c for HPV-16 E6 (Santa Cruz Biotechnology); and the murine monoclonal antibody, TVG710Y, speci®c for HPV-16 E7 (Santa Cruz Biotechnology). Chemicals Lactacystin (Calbiochem-Novabiochem Corp.) was dissolved in DMSO and added to the culture media for 2 h. The ®nal concentration of lactacystin and DMSO were 5 mM and 0.1%, respectively.
Acknowledgements We would like to thank Marcel A Baluda and Bradley Ackerson for critical reading of this manuscript. This work was, in part, supported by grants DE11229 and DE10598 from the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA.
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