p53 mutations and human papillomavirus DNA in oral squamous cell carcinoma: correlation with apoptosis.

Forty-two oral squamous cell carcinomas (SCCs) were analysed for p53 mutations and human papillomavirus (HPV) infection to examine the prevalency of these factors and correlation with apoptotic index (AI; number of apoptotic cells per 100 tumour cells) of the tumour tissue. In polymerase chain reaction (PCR)-Southern blot analysis, HPV DNAs were detected from 22 out of 42 SCCs (52%) with predominance of HPV-16 (68%). p53 mutations in exons 5-8, screened by nested PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, were observed in 16 of 42 tumours (38%). The state of the p53 gene did not show any correlation with HPV infection. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labelling (TUNEL) method was used for detection of apoptotic cells. The mean AI was 2.35, ranging from 0.31 to 6.63. SCCs associated with p53 mutation had significantly lower AI than those without p53 mutation (P < 0.01), whereas no difference in AI was found between SCCs with and without HPV infection. The results of this study confirmed that HPV infection and/or p53 mutations are implicated, but are not mutually exclusive events, in carcinogenesis of oral SCC and also showed that decrease in apoptosis is more closely related to p53 mutation than HPV infection.

Squamous cell carcinoma (SCC) is the most common malignant neoplasm of the oral mucosa. representing more than 90% of intraoral malignant tumours. Tobacco and alcohol use. viral infections. nutritional deficiencv and dietary customs have all been implicated in the aetiology of head and neck cancer (Regezi et al. 1993). W'hereas the epidemiology has been w-ell described so far. the molecular steps involsed in the pathogenesis of these common neoplasms are poorly understood. The role of human papillomas-irus (HPV) in the development of anogenital cancers has been widely studied. and current evidence shows that HPV infection is necessars for the desvelopment of most cervical cancers (zur Hausen. 1994). Approximately 80-90% of cervical carcinomas contain HPV DNA. and the predominant or high-risk types appear to be HPV-16. -18 and -33 (Yoshikawa et al. 1991). HPV E6 and E7 proteins. consistentlv expressed in HPV-transformed and HPVpositive tumours. can exert their oncogenic potential bv inactivating the products of the p53 tumour-suppressor cene (Lemine. 1990: zur Hausen. 1994) and the retimoblastoma (Rb) grene (Dy-son et al. 1989). In oral malignant lesions. the state of HPV infection has been reported to be as high as 76%c (Snijders et al. 1994). although there are still conflicting results in infection rate and role of HPV in oral carcinorenesis. Received 26 September 1997Revised 5 January 1998Accepted 16 January 1998 Correspondence to: JY Koh. Department of Oral Pathology. School of Dentistry. Chonbuk National Universrty.  Duckjlin-dong. Chonju 560-756. Republic of Korea p53 plays an important role in the maintenance of genomic integrits through the induction of cell growth arrest or apoptosis follossin, DNA damage (Weinberg. 1991: Smith et al. 1995. The loss of a functional tumour-suppressor protein such as p53 has been implicated in the aetiologand progression of a variety of human tumours (Levine. 1990: Hollstein et al. 1991a. Indeed. p53 mutation is the most frequently detected genetic alteration in human cancers. particularly in tobacco-related tumours of the lung. oesophagus and oral cavity (Hollstein et al. 1991b). The loss of activity of the wild-type p53 protein can be achiesed bv two different mechanisms: either by a mutation of the p53 gene (Crook et al. 1991 ) or by binding to the HPV encoded E6 protein (Scheffner et al. 1990). In oral cancer. both mechanisms of inactivating p53 may play a role in carcinogenesis. Several studies. analysing the relationship betu-een p53 mutations and HPV infection in malignant head and neck tumours. have shown high frequencies of p53 mutations in HPVpositixe oral cancers (Brachman et al. 1992: Barten et al. 1995. Apoptosis. or programmed cell death. is an essential mechanism that regulates cell loss in tissue modellinc for vertebrate deselopment with apparent differences from necrosis. This process is a unique feature of multicellular organisms that enables continuous renewal of tissue by cell division while maintaining the steadvstate lesvel of the various histological compartments under tight control (Raff. 1992). Regulation of the process leading to a phy siological cell death involhves expression of sexeral proto-oncogenes or tumour-suppressor genes. such as c-mvc. bcl-2 and p53 (Clarke et al. 1993). Failure of tumour cells to undergo apoptosis can result in uncontrolled accumulation of cells (Kerr et al. 1994).
Morphologically. apoptosis is characterized by nuclear chromatin condensation and budding, of the cell. with formation of  or2anelles. which are phagocvtosed and digested by nearby resident cells (Kerr et al. 1972: Wxllie et al. 1984. Because of the short duration of morphological changes and seemingly low incidence. it is difficult to detect apoptosis in routine histological sections. Such difficulties can be o-ercome by using the terminal deoxx-nucleotidx 1 transferase (TdT)-mediated dUITP-biotin nick end labelling (TUNEL) method. which detects apoptosis by in situ labelling, of DNA breaks in indi-idual nuclei in tissue sections processed throurh the routine procedures of histopathologx.
Recently. there has been growing interest in apoptosis in relation to histopathological differentiation. tumour progression and its prognostic value (Kasagi et al. 1994: Tormainen et al. 1995. There is. howexer. no report axailable so far on the significance of apoptosis in oral cancer in relation to p53 mutation and/or HPV infection. Therefore. we exaluated the pre-alence of HPV infection and p53 mutations in individuals w ith malignant oral lesions and examined the relationship of these factors w-ith apoptotic index (Al) of the tumour tissue.

MATERIALS AND METHODS
Forty-two cases of oral SCC were collected from the 1989-1996 pathological files of Chonbuk National University Hospital and examined by light microscopy using sections stained routineiv with haematoxy lin and eosin (H&E). Tissue sections containing more than 20% tumour tissue w-ere used: verrucous carcinoma w-as not included in this studv.
For visualization of the hybridization results. membranes were exposed to Agfa X-ray film with an intensifying screen at -70'C for 18-24 h.

Analysis of p53 mutations by nested PCR-SSCP
Exons 5-8 of the p53 gene were analysed for the presence of mutations using nested PCR amplification followed by SSCP analysis (Orita et al. 1989). The oligonucleotide primers for exons 5-6. 5. 6. 7. 8 (outer). and 8 (inner) were prepared according to the sequence published by Buchman et al (1988). In the present study. the nested PCR method wvas used to overcome a difficulty in amplification of DNA extracted from formalin-fixed. paraffinembedded tissue. Briefly. an aliquot of 100-200 ng of genomic DNA was amplified in a v-olume of 10 gl containing 50 nm\i potassium chloride. 10 m\ Tris-HCl (pH 8.3). 1.5 mn\ magnesium chloride. 0.1 nmn of each dNTP. 10 pmol of each first set primer. 0.25 units of Taq DNA polymerase. The outer nested PCR amplification was performed for 30 cycles with denaturation for 1 min at 95CC. annealing for 1 min at each optimal temperature (55-C for exon 8 outer. 58 C for exons 5. 5-6. 7 and 8 inner. 60-C for exon 6) and extension for 1 min at 72CC. An aliquot of 0.5 gl of the product of this reaction was transferred to a second reaction mixture containing the same medium as before. but with the inner pair of nested primers labelled with [r-'P]ATP A further 25 cycles were carried out under the same conditions as above. Positise controls from normal oral mucosa and blanknegative controls Aw ere included in each reaction. A 2-j1-aliquot of PCR products was diluted 10-fold with buffer containin, 95%c formamide. 10 m-EDTA. 0.05% bromophenol blue and 0.05% xylene cvanol. then heat-denatured at 98^C for 5 min followed by cooling on ice. An aliquot of 2-4 gl of this p53, HPV and apoptosis in oral carcinoma 357 mixture was loaded into a 6%c non-denaturing polyacrylamide gel A containing 5% glycerol and run at constant power of 20 W for 6-8 h under cooling with a fan at room temperature. Electrophoresis was performed by using a sequencing-type apparatus (Bio-Rad Laboratories. USA) using 0.5 x TBE as rumning buffer. Gels were dried on filter paper and exposed to Agfa X-ray film with an intensifying screen at -70°C for 12-24 h. All the samples were subjected_ to duplicated or triplicated reactions.

Detection of apoptosis by the TUNEL mrethod
The TUNEL method was perforned according to the description of Gavnreli et al (1992). with slight modification. Briefly. after deparaffinization and hydration. sections were digested with 20 jg ml-' proteinase K at room temperature for 15 mum and endogenous peroxidase was inactivated by covering with 2 hydrogen peroxide for 5 min. Tissue sections were immersed in terminal deoxvnucleotidvl transferase (TdT) buffer (30 n-Xi Tris-HCl. pH 7.2. 140 iNt-sodium cacodylate. I inst cobalt chloride) at room temperature for 1O mn. and then incubated with 0.3 units pl-' TdT (Trevigen. i-Maryland. USA) and 0.01 nmol 1-' biotin-Il-dUTP in TdT buffer at 37°C for 60 mm. After washing wvith PBS. streptavidin peroxidase was applied and finally the sections were visualized using diaminobenzidine (DAB) and counterstained with 1% methyl green. Normal lymph node tissues and reactions without TdT were used as positive and negative controls respectively.

Al
The slide was randomly moved and five fields were selected and photographed for each case (magnification x 400). All TUNEL signal positive nuclei from at least 1000 tumour cells were then counted. Apoptotic labelling indices were calculated as number of positive cells per 100 tumour cells. c Statistical analysis O x Statistical analysis was performed using the unpaired Student's ttest. except for the correlations between HPV infection and p53 mutations. which were calculated using the chi-square test. A Pvalue below 0.05 was considered significant. Data were presented as mean ±s.e.

RESULTS
All 42 samples were histopathologically diagnosed as SCCs at various grades of differentiation. The majonity of them were well differentiated (69%). and the rest were moderately (21%) or poorly differentiated (10%). DNA samples that gave no amplification products for p53 were considered inadequate and excluded from this studv.  (1998) 78(3), 354-359 (38%). These 16 tumours had mutations in each of the analysed exons as follows: exon 5 in six tumours, exon 6 in five tumours, exon 7 in four tumours and exon 8 in four tumours. Among them. two cases showed combined mutations in exonS and exon 6, one case in exon 6 and exon 8 (Table 2, Figure 2). p53 mutations were present in 10 of 22 HPV-positive carcinomas (45%) and six of 20 HPV-negative tumours (30%). Fourteen carcinomas of 42 samples (33%) had neither HPV DNA nor p53 alterations. The state of the p53 gene did not show any correlation with HPV infection.

Ai
Cells undergoing apoptosis showed condensation of nuclear chromatin, nuclear fragnts (apoptotic bodies) and loss of cell-cell contacts in routine H&E sections. TUNEL signals were randomly distributed in tumour tissues. They were detected not only in tumour cells showing chromatin condensation but also in morphologically viable cells (non-pyknotic cells) at the start of apoptosis, as identified by distinct nuclear staining (Figure 3). Nonneoplastic epithelium was available adjacent to the tumour tissues from 27 of 42 samples. TUNEL-positive cells were rarely observed in the adjacent epithelium except in the most superficial layer. which showed not only nuclear but also cytoplasmic staining. It was difficult to determine whether these signals were specific or not.
In 42 oral SCCs. the Al was averaged to be 2.35 ± 0.23 (s.e.), ranging from 0.31 to 6.63. The mean Al was 2.72 ± 0.28 in 16 tumour samples with wild-type p53 and 1.73 ± 0.34 in those with p53 mutation. This difference was statistically significant (P < 0.01). There was, however, no difference in Als between tumours with and without HPV infection (Table 3).

DISCUSSION
HPV DNAs have been found in various locations in the human body and there is some certainty that HPVs play an important role in carcinogenesis, especially in genital lesions (Herrington, 1995). As oral mucosa is covered by squamous epithelium that resembles cervical epithelium, it is important to investigate the relationship between HPV infection and oral cancers. Many studies have reported frequent association of the HPV in oral cancers, although the exact nature of its relationship to oral carcinogenesis remains obscure (Brachman et al, 1992;Barten et al, 1995).
We examined 42 oral SCCs for the presence of HPV DNA using PCR-Southem blot analysis with primers and probes specific for HPV-16, -18 and -33, which have been detected frequently in cervical carcinomas. HPV DNA sequences were identified in 22 of 42 samples (52%) with predominance of HPV-16 (15/22). This finding is consistent with previous reports that have used PCRbased methods to examine oral cancer tissues (Kiyabu et al. 1989;Shindoh et al. 1992). The results of the present study indicate that HPV infections are important but may not be sufficient for the progression to malignancies and that synergistic actions with other carcinogenic agents may be required. There is also an argument against a singular role for HPV in oral carcinomas as some authors reported high prevalence of HPV infection in a normal control population (Jenison et al, 1990;Jalal et al. 1992). The possibility cannot be excluded that HPV from adjacent normal or dysplastic epithelium may have contributed. As compared with the epithelium of the uterine cervix, the oral mucosa is continuously exposed to a number of environmental carcinogens. including tobacco and alcohol. Tlese factors may act synergistically with HPV. leading to the development of carcinomas (Mao et al. 1996).
The p53 gene and its product have been studied extensively ever since it became clear that more than 50% of human cancers contain mutations in this gene, including carcinomas of the colon, lung and breast (Levine, 1990;Hollstein et al, 1991a). p53 proteins encoded by mutant alleles are often more stable than wildtype p53. resulting in a dramatic increase in p53 expression and inactivation of wild-type p53 by a dominant-negative mechanism (Levine, 1990;Weinberg, 1991). In addition to genetic change. an altemative mechanism for the functional inactivation of p53 is the formation of protein complexes with cellular proteins or viral oncoproteins. HPV E6 and E7 proteins are consistently expressed in HPV-transformed cells and in HPV-positive tumours, and the E6 protein fonms a complex with the p53 protein, resulting in degradation of p53 (Scheffner et al, 1990;zur Hausen, 1994). This targeted degradation of p53 by the E6 proteins would account for the lowered levels of p53 protein found in HPV-immortalized squamous epithelial cell lines (Scheffner et al, 1991).
The state of the p53 gene was investigated by SSCP analysis of PCR products, which is a fast and sensitive method for detection of sequence changes including single-base substitutions (Orita et al. 1989). Mutational analysis of the p53 gene was restricted to exons 5-8, where over 90% of the p53 mutations have been found in other human malignancies (Hollstein et al. 1991a). In this study.
p53 gene mutations were detected in 16 of the 42 oral SCCs (38%). This prevalence of mutations is similar to that reported in studies of invasive head and neck carcinomas: 24% (Chiba et al, 1996) and 42% (Mao et al, 1996).
An interesting observation in this study was the frequent p53 mutations in HPV-positive oral carcinomas (10/22), suggesting that HPV and p53 mutations may not be mutually exclusive events. This is in contrast to the situation in cervical carcinomas, in which mutations of the p53 gene appear to be rare in cases associated with HPV infection, but common in malignancies devoid of HPV infection (Crook et al, 1991;Park et al. 1994). As co-expression of high-risk E6 protein together with wild-type p53 protein can result in the same phenotypic effect as mutation of the p53 gene, inactivation of the p53 gene by both mutation and binding to the HPV oncoprotein E6 might seem unnecessary. But these two factors may act on the same cell and cooperate with each other.
The presence of both HPV DNA and p53 mutations in the same tumours in the present study provides some evidence for such cooperation.
Apoptosis is a basic biological phenomenon of critical importance in the regulation of the cell population in situations as diverse as embryonic growth and modelling, hormone-induced organ involution and neoplasia (Kerr et al. 1972). We detected apoptotic cells in oral SCCs by using the TUNEL method. Intense TIJNEL signals were frequently observed in nuclei of tumour cells showing chromatin condensation, and occasionally even in ordinary, non-pyknotic nuclei of tumour cells. Grasl-Kraupp et al (1995) reported that the TUNEL assay fails to distinguish apoptosis from necrosis and should not be considered as a specific method for detecting apoptosis. In the present study, the possibility was excluded by comparing the TllNEL-positive cells with histological findings, including inflammation in H&E-stained sections.
In the 42 oral SCCs, the mean Al ( p53, HPV and apoptosis in oral carcinorma 359 various locations, which ranged from 1.5 to 10.9 (Kasagi et al, 1994;Shoji et al, 1996;Tatebe et al, 1996). In relation to p53 status, SCCs associated with p53 mutation had significantly lower Al than those with wild-type p53 (Table 3). These results suggest that wild-type p53 may promote apoptosis and that mutant p53 might be involved in the inhibition of apoptosis in oral SCCs. No significant difference in Als was found between tumours with and without HPV infection. But it could not rule out the possibility that HPV E6 protein may play a role in apoptosis as Als between tumours with and without HPV infection were hampered by the frequent p53 mutations in our study. Investigation of the HPVpositive and -negative tumours without p53 mutation, in a large series, will be required to determine whether this notion is correct. There is increasing evidence that apoptosis may also be involved in the progression of cancer, although conflicting results have been reported regarding AI and patient prognosis (Kasagi et al, 1994;Tormanen et al, 1995). Theoretically, an increase in apoptosis may result in tumour regression, but some authors have proposed that apoptosis may reflect not only cell loss but also the proliferative activity of the cancer (Tatebe et al, 1996). Additional studies will determine if detection of apoptosis can be used as a prognostic parameter for the oral cancer.
In conclusion, this study confirms that HPV infection and/or p53 mutations are implicated, but are not mutually exclusive events, in carcinogenesis of oral SCC. The results also show that decrease in apoptosis in oral cancer is more closely related to p53 mutation than HPV infection. Further study is necessary to define the exact role of apoptosis in differentiation and progression of carcinoma and prognostic value of Al in oral cancers.