Japanese Journal of Clinical Oncology 34:176-183 (2004)
© 2004 Foundation for Promotion of Cancer Research
Involvement of Viral and Chemical Factors with Oral Cancer in Taiwan
1 Department of Medical Research and 2 Department of Obstetrics and Gynecology, Show Chwan Memorial Hospital, Changhua, Taiwan, 3 Department of Surgery and 4 Department of Pathology, Chung Shan Medical University Hospital and 5 School of Medical Technology, Chung Shan Medical University, Taichung, Taiwan, ROC
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
Background: The association between oral squamous cell carcinoma (OSCC) and viral and chemical factors is uncertain. Therefore the correlation of viral and chemical factors with oral cancer in Taiwan was investigated.
Methods: Thirty-seven paraffin-embedded oral cancer biopsies and 36 normal oral tissue specimens were examined by the polymerase chain reaction method for six viruses: HPV, CMV, EBV, HSV-1, HSV-2 and HHV-8. To elucidate the role of arecoline in the oncogenesis of oral cancer, human buccal fibroblasts, oral submucosal fibroblasts and three cancer cell lines KB, GNM and TSCCa were used for MTT cytotoxity assay and flow cytometry DNA content analysis.
Results: Two (5.4%) HSV-1-positive and four (10.8%) HPV-positive cases were recognized in oral cancer biopsies. Among the four HPV-positive tissues, two were further typed as HPV-16, one was identified as HPV-18- and HSV-1-positive; and one contained both HPV-16 and HPV-18. One sample presented HSV-1 only. Arecoline, at a concentration lower than 0.8 µg/ml, increased cell growth (all cell types); at higher concentrations (25–400 µg/ml) it was cytotoxic. The cell cycle was demonstrated to be altered either by low or high concentrations of arecoline treatment, depending on the cells treated.
Conclusions: The data demonstrated that HPV, HSV-1 and betel quid chewing were significantly associated with OSCC, but HSV-2, CMV, EBV and HHV-8 were not. We suggest that the most determinative factor for oral cancer may be chemical in nature rather than viral infection.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
Studies have shown that during the last decade in Taiwan, oral squamous cell carcinoma (OSCC) has ranked fifth among cancer-related mortality. More than 1000 people have died of OSCC every year. About 90% of patients were male. Among the Taiwanese population, an association between OSCC and viral and chemical factors, if any, is uncertain.
Human papillomavirus (HPV) is classified into the Papillomaviridae family. It has been known for many years that HPV infection plays an important role in the carcinogenesis of cervical cancer (1). HPV has also been investigated in patients with oral cancer (2). HPV can infect the squamous epithelium of both the uterine cervix and the oral mucosa, leading to the formation of benign or malignant tumors (3). Herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein–Barr virus (EBV) and human herpes virus (HHV)-8 are members of the Herpesviridae family. HSV-1 infection occurs mainly in the mouth and lips, while HSV-2 infection usually occurs in the genital tract. However, there is a great deal of overlap between the epidemiology and clinical manifestations of infection. HHV-8 is considered to be causally associated with Kaposi’s sarcoma (4). CMV is a common human pathogen and is spread mainly by either a sexual or transfusion route. It was suggested to be associated with cervical cancer (5). EBV is the causative agent of infectious mononucleosis (IM) and is closely associated with Burkitt’s lymphoma (BL), nasopharyngeal carcinoma (NPC) and opportunistic B-cell lymphoma in immunocompromised hosts (6–8).
Betel nut, as analyzed by HPLC, contains about 5.5% arecoline (9). Bacterial and cellular experiments suggested arecoline is a mutagen (10), which stimulates the proliferation of fibroblasts and collagen synthesis (11). The products of N-nitrosation of arecoline, i.e., 3-methylnitrosamino-propionitrile (MNPN) and 3-methylnitrosamino-propion aldehyde (MNPA) are suggested to be powerful carcinogens in rats (12). In different studies, arecoline showed different effects on different oral cells. Between 0 and 10 µg/ml of arecoline increased the viability of normal oral submucous fibrosis tissue (13). At concentrations of 0.2–2 µM, arecoline increased micronucleated CHO (Chinese hamster ovary) cells and prolonged the cell cycle (14). However, 50–150 µg/ml arecoline inhibited oral mucosal fibroblasts and gingival fibroblast growth and was cytocidal at 300–500 µg/ml (9,15,16).
Thus, the purpose of this study was to investigate the correlation of virus and chemical factors with oral cancers in mid-Taiwan. We studied HPV, CMV, EBV, HSV-1, HSV-2 and HHV-8 to investigate the different factors involved in oncogenesis of oral cancer. Moreover, the effects of different dosages of arecoline on the oncogenesis of different oral cells such as normal buccal fibroblasts (BF), oral submucosal fibroblasts (OSF) and three different oral cancer cell lines, GNM (neck metastasis of gingival carcinoma), KB (epidermal cells of the mouth) and TSCCa (tongue squamous cell carcinoma), were also investigated. The present study is significant in that it may allow delineation of chemical and/or viral etiological factors in the development of oral cancers in the mid-region of Taiwan.
|
SUBJECTS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
Samples
Thirty-seven paraffin-embedded OSCC biopsies and 36 specimens of control oral mucosa tissue were collected. The tissues of the control group included normal or inflammatory mucosa obtained from negative biopsy, teeth extraction and excision of pyogenic granuloma, fibrous hyperplasia and leukoplakia. All the samples were collected at Chung Shan Medical University Hospital, Taichung City and Show Chwan Memorial Hospital, Changhua City between 1997 and 2002. Both hospitals are located in mid-Taiwan. Patient details of gender, age, past histories of betel quid chewing and medical illness were obtained from medical charts or by personal inquiry. Three (8.1%) patients with OSCC had no or occasional betel quid intake and the other 34 (91.9%) patients had habitual intake of one or more pack/day of betel quid for more than 5 years. Only four (11.1%) of the 36 control patients had habitual intake (less than one pack/day) for 3, 5 and 6 years, respectively, while the other 32 (88.9%) patients had no betel quid chewing history.
DNA Extraction and PCR Assay
DNA was extracted according to the traditional phenol/chloroform method (17). For the nine frozen cervical cancer specimens, 50 mg of each was homogenized in 1.5-ml Eppendorff tubes. After treatment with proteinase K at 56°C for 60 min, DNA was extracted using the phenol/chloroform procedure and purified by ethanol precipitation. For the PCR assay, the dried DNA was resuspended with deionized water to make a final concentration of 50 ng/µl. Paraffin-embedded specimens were dewaxed with xylene and ethanol, followed by DNA extraction. Target DNA was amplified in a 20-µl reaction mixture containing 2 µl of sample DNA, 10x Taq polymerase buffer (20 mM KCl, 10 mM Tris–HCl, 1.5 mM MgCl2, 0.1% Triton-X100), 2.5 U of Taq polymerase (Promega, Madison, WI), 200 pmol of each primer, 2.5 mM of deoxyribonucleoside triphosphate (dNTP) and 50 µl of mineral oil. Thermal cycles were performed in a programmable PCR thermal cycler. Primers and thermal cycler programs for identification of each of the viruses (CMV, EBV, HSV-1, HSV-2, HHV-8 and HPV) are listed in Table 1. Nine pairs of primers were used for HPV typing. ß-Globin, HPV18-plasmid, CMV, EBV, HSV-1, HSV-2 and HHV-8 positive clinical samples were also used as controls. PCR products were examined by direct agarose gel electrophoresis stained with ethidium bromide. Each sample was tested three times by PCR.
|
Cells and Cell Culture
The oral cells tested in this study were kindly provided and characterized (18) by Dr Ming-Yung Chou and Dr Yu-Chao Chang, Department of Dentistry, Chung Shan Medical University Hospital. The two strains of BF cells were derived from healthy individuals that did not drink alcohol, smoke tobacco or chew areca nuts. The three strains of OSF cells were derived from alcoholic individuals that habitually smoked tobacco and chewed areca nuts and were characterized as pre-cancer cells. The three cancer cell lines were GNM, KB and TSCCa. Human BF, GNM, KB and TSCCa cells were cultured in Dulbecco’s modified Eagle’s medium containing 1% penicillin/streptomycin and 10% fetal bovine serum. Under these conditions, the doubling time of OSF, GNM, KB and TSCCa cell lines was between 31 and 34 h. The doubling time of BF was 62 h. Experiments used BF cells from early passages (3–8 passages).
3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyl Tetrazolium Bromide Assay
A microculture tetrazolium (MTT) colorimetric assay was developed to monitor mammalian cell survival and proliferation. The MTT assay measured dehydrogenase activity, as described by Mosmann (19) with minor modification (18). In brief, aliquots of 1 x 103 cells/ml, in triplicate, were plated into a series of 96-well microplates (100 µl culture/well) for overnight incubation at 37°C in 5% CO2. Then, 0.4–400 µg/ml of arecoline (Sigma, St Louis, MO) was dispensed within appropriate wells (in triplicate) for 12, 24 or 48 h of incubation. After arecoline treatment, the supernatant was discarded and replaced with 100 µl of MTT (Sigma, final concentration, 0.5 mg/ml) for 4 h of incubation. Then, 100 µl dimethyl sulfoxide (DMSO) was added and mixed thoroughly for absorbance readings at 550 nm. Cell viability % = (absorbance of arecoline-treated cells/absorbance of negative cells) x 100.
Cell Cycle Analysis
Samples of 4 x 105 cells were incubated in 6-cm Petri dishes for overnight cell cycle analysis. BF, OSF, KB, GNM and TSCCa cells were treated with different amounts of arecoline, in triplicate, for 48 h. After treatment, the cells were trypsinized and centrifuged at 1200 r.p.m. for 5 min before being fixed in 80% ethanol at 4°C overnight. The cells were then treated for 30 min at 37°C in the dark with 1 ml of prodium iodide (PI) solution (50 µg of PI/ml, 0.1% sodium citrate, 0.2% NP-40, 0.25 mg RNase/ml). Then, the cells (20 000 events collected from each) were ready for flow cytometry (Becton Dickinson Fasscalibur, San Jose, CA) analysis. PI-negative (live) cells at the G0/G1, S and G2/M fractions were counted and modeled with the Cellfit cell cycle analysis program (Becton Dickinson).
Statistical Analyses
The presence of viral DNA in oral cancer tissues was analyzed by binominal test; the relationship of cytotoxicity of arecoline with dose and time was analyzed by two-way ANOVA analysis; the DNA content of different cells, as assayed using a fluorescence-activated cell sorter (FACS), was analyzed by simple linear regression. Comparison of the positivities of viral factors and betel quid chewing in the oral cancer group was analyzed by Fisher’s exact test. P value < 0.05 was taken to be significant in all analyses.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
Thirty-seven paraffin-embedded OSCC specimens and 36 control oral tissues specimens, for a total of 73 specimens, were collected for viral DNA study (HPV, CMV, EBV, HSV-1, HSV-2 and HHV-8). Among the 37 paraffin-embedded oral cancer specimens only two had HSV-1 (5.4%) detected and four HPV-positive (10.8%) samples were found by PCR (Fig. 1a and b). Among the 36 specimens of normal oral tissue, no viral DNA was detected in redundant PCR tests. Analyzed by binominal test, the presence of both HSV-1 in two and HPV in four oral cancer tissues was considered significant (P < 0.05). Of the five HSV-1- or HPV-positive samples, the two were further type-identified as HPV-16, one was identified as HPV-18, one contained both HPV-16 and HPV-18, one of the HSV-1-positive samples showed HPV-18 as well, whereas another showed HSV-1 only. In order to rule out the possibility that there was too small an amount of HPV in the specimens to detect, differing amounts of the HPV-plasmid DNA were serial diluted and detected by PCR. The sensitivity test suggested that between 0.1 ng and 6.4 fg of viral DNA was detectable by 40 cycles of PCR (Fig. 1d). The results of viral DNA detection and the history of betel quid chewing in patients with OSCC and controls are tabulated in Table 2. Thirty-four (91.9%) out of 37 patients had taken betel quid, compared with four (11.1%) of the 36 controls. The data indicated that HPV, HSV and betel quid chewing were significantly associated with OSCC (P < 0.05), but CMV, EBV, HSV-2 and HHV-8 were not (P > 0.05).
|
|
As areca nuts contains large amounts of arecoline, the susceptibility and effects of arecoline on the cell cycles of human BF, OSF and three oral tumor cell lines (KB, GNM and TSCCa) treated with arecoline were investigated to study the effects of chemical treatment. Different oral cells were used with different amounts of arecoline treatment, and analyzed using the MTT assay and flow cytometry. Two strains of BF, three strains of OSF and three cancer cell lines (KB, GNM and TSCCa) were treated with different amounts of arecoline for different time periods and tested using the MTT assay for cell viability (Fig. 2). The results revealed that 25–400 µg/ml (high concentration) of arecoline treatment for 12, 24 or 48 h were significantly cytotoxic (P < 0.05) to different percentages of BF, OSF, KB, GNM and TSCCa cells. Data analysis by two-way ANOVA demonstrated that the cytotoxicity of arecoline was related to dose and time. On the other hand, 0.4–0.8 µg/ml (low concentration) of arecoline treatment for 12, 24 or 48 h had significantly different effects on these cells. Cell viability was significantly increased by the low concentration of arecoline treatment (P < 0.05) but the proliferative effect was not related to dose and time (P > 0.05). In addition, the cytotoxic and proliferative effects of arecoline in high or low doses, respectively, were consistent on primary (BF), pre-cancer (OSF) and cancer cells (KB, GNM, TSCCa).
|
Fig. 3 demonstrates the DNA content of different oral cells as assayed using FACS, and the data were analyzed by simple linear regression. After treatment for 48 h, neither live BF nor OSF cell cycles were significantly affected by 25–400 µg/ml (high concentration) of arecoline (Fig. 3a and c). On the other hand, at 0.4–0.8 µg/ml (low concentration, Fig. 3b and d), S phase was increased, while G0G1 phase was decreased, and these events correlated with increasing dosage from 0.4 to 0.8 µg/ml of arecoline for both BF and OSF cells. For cancer cells, the live KB cell cycle was not significantly affected at 25–400 µg/ml of arecoline treatment (Fig. 3e), but S phase was increased and G0G1 phase was decreased and correlated with increasing dosages at 0.4–0.8 µg/ml of arecoline (Fig. 3f). The live GNM cell cycle was not significantly affected at 0.4–0.8 µg/ml of arecoline (Fig. 3h), but S phase was increased and G0G1 phase was decreased, correlating with increasing dosages of 25–400 µg/ml of arecoline (Fig. 3g). The live TSCCa cell cycle was not significant affected at 0.4–0.8 µg/ml of arecoline (Fig. 3j), but G2M phase was increased and S phase was decreased, correlating with increasing dosages of 25–400 µg/ml high concentration of arecoline treatment (Fig. 3i). These results revealed that different cell cycle phases were affected by high or low concentrations of arecoline treatment on these different cells. Thus, the cytotoxic and proliferative effects by high or low concentrations of arecoline depended on the cell type.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
In the present study, specimens used for analysis were formalin-treated and paraffin-embedded. It was of concern that formalin treatment and paraffin-embedding storage might have affected the DNA stability. As noted in the Results, however, only two cases of HSV-1 and four HPV-positive were detected among 37 oral specimens. The low correlation results between oral cancer and HPV, noted in the present study, is different from results reported by our colleagues, Chen et al. (20), revealed 85.7 and 71.4% of HPV-16 and HPV-18 present in OSCC cases, respectively, by in situ PCR ISH (in situ hybridization) detection. Although race and geographical area were similar for the samples, the difference in results between the two studies may be related to other factors, including clinical condition of the patients and sample analysis technique. The technique and primers used by Chen et al. were also different from that used in the present study. As our sensitivity test result revealed that between 0.1 ng and 6.4 fg of viral DNA was detectable by 40 cycles of PCR, it suggests that PCR is one of the most sensitive methods of virus identification. In this study, the presence of viral DNA was thus identified using the PCR method. Recently, biotinyl-tyramide-based ISH methodology was used to investigate the prevalence of HPV integration in cervical intraepithelial neoplasia (21). This method was considered sensitive enough to detect integrated viral DNA by punctuate signals within the nucleus, and episomal viral DNA by a diffuse signal throughout the nucleus. For our HPV PCR, the selected primers were within gene late 1, which is a conserved region. In order to rule out the possibility that the HPV late 1 region did not integrate into the cellular genomic DNA, and therefore could not be detected by PCR, the DNA of the oral specimens was treated with topoisomerase I to loosen supercoiled DNA according to the method described by Laghi et al. (22). However, still no difference of positive/negative was detected (data not shown).
According to clinical and epidemiological observations in Taiwan, most patients with oral cancer are addicted to areca nut chewing (2,23). It has been reported that the oral level of arecoline in areca nut chewers was low and below 140 µg/ml (24). The low level of arecoline might stimulate the growth of oral tissue, causing fibrosis of the oral mucous membrane and promotion of oral pre-cancer formation. Our results demonstrated that low concentrations of arecoline (0.4–0.8 µg/ml) in culture conditions stimulated cell growth and DNA synthesis, whereas high concentrations (25–400 µg/ml) inhibited cell growth. In the present study, depending on which cells were treated, the cell cycle phases were affected by low or high concentrations of arecoline treatment. These results suggest the possibility that oral cells may have been transformed under in vitro conditions by low amounts of arecoline.
Although cervical and oral areas are composed of similar mucous tissue, both the oncogenic factors and underlying oncogenic mechanisms may be quite different. Epidemiological and molecular studies suggest that cervical infection by certain types of HPV is a precursor event in the genesis of cervical neoplasia (25). Walboomers et al. (26) reported a high association between HPV and cervical cancer: up to 99.7%. On the other hand, viral factors, especially HPV, appear to be less closely related to oral cancer (10.8% association in this study). Since one of the HSV-1-positive sample showed HPV DNA as well, it may also suggest that HSV-1 could be a cofactor in the oncogenesis of oral cancer or could infect cancer tissues opportunistically. Other viruses such as HSV-2 and CMV, EBV and HHV-8 are not determinative and might not be involved in the oncogenic processes directly. Therefore, the possibility of oral cancer caused by virus infection, except for HPV and HSV-1, is very low. Because of the possibility of frequent exposure, oral tissues are more likely to be stimulated by chemical factors than cervical tissues. The in vitro results of arecoline treatment in the present study suggest the possibility of oral cell transformation by chemical factors. Analyzed by Fisher’s exact test, the positivity (5.4–10.8%) of viral factors is lower than the positivity (91.9%) of betel quid chewing in the oral cancer group. Comparing the relatedness statistically, one may suggest that chemical factors could play more significant roles in oral cancer oncogenesis than viral factors. Therefore, we proposed that the correlation between viral infections and oral cancer in Taiwan may be very low. The most determinative factor for oral cancer might be chemical rather than viral in nature.
|
Acknowledgments |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
The authors are indebted to Dr Ming-Yung Chou and Dr Yu-Chao Chang, Department of Dentistry, Chung Shan Medical University Hospital, Taichung, Taiwan for the generous gift of BF, OSF, GNM, KB and TSCCa cells. We thank Professor Ravindra M. Shah, Faculty of Dentistry, University of British Columbia, for careful proofreading. This study was funded by a grant (CSMC88-OM-B-035) from the Chung Shan Medical University, Taichung, Taiwan, ROC.
|
FOOTNOTES |
|---|
+ For reprints and all correspondence: Chi-Chiang Yang, School of Medical Technology, Chung Shan Medical University, 110, Section 1, Chien-Kuo North Road, Taichung, Taiwan 402, ROC. E-mail: cyang{at}csmu.edu.tw
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
1 Morrison EA, Ho GF, Vermund SH, Goldberg GL, Kadish AS, Kelly KF, Burk RD. Human papillomavirus infection and other risk factors for cervical neoplasia: a case-control study. Int J Cancer 1991;49:6–13.[Web of Science][Medline]
2 Chang KW, Chang CS, Lai KS, Chou MJ, Choo KB. High prevalence of human papillomavirus infection and possible association with betel quid chewing and smoking in oral epidermoid carcinomas in Taiwan. J Med Virol 1989;28:57–61.[Web of Science][Medline]
3 Portugal LG, Goldenberg JD, Wenig BL, Ferrer KT, Nodzenski E, Sabnani JB, Javier C, Weichselbaum RR, Vokes EE. Human papillomavirus expression and p53 gene mutations in squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 1997;123:1230–4.
4 Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J, Knowles DM, Moore PS. Identification of herpes virus-like DNA. Science 1994;266:1865–9.
5 Odida M, Schmauz R. Cervical cancer and cytomegalovirus. East African Med J 1996;73:810–2.[Web of Science][Medline]
6 Henle G, Henle W, Diehl V. Relation of Burkitt’s tumor-associated herpes-type virus to infectious mononucleosis. Proc Natl Acad Sci USA 1968;59:94–101.
7 zur Hausen H, Schulte-Holthausen H, Klein G, Henle W, Henle G, Glifford P, Santesson L. EB-virus DNA in biopsies of Burkitt tumors and aplastic carcinomas of the nasopharynx. Nature 1970;228:1056–7.[CrossRef][Medline]
8 Hamilton-Doutoit SJ, Pallesen G, Franzmann MB, Karkov J, Black F, Skinhøj P, Pedersen C. AIDS related lymphomas. Histopathology, immunophenotype and association with Epstein–Barr virus as demonstrated by in situ nucleic acid hybridization. Am J Pathol 1991;138:149–63.[Abstract]
9 van Wyk CW, Oliver A, de Miranda CM, van der Bijl P, Grobler-Rabie AF. Observations on the effects of areca nut extracts on oral fibroblast proliferation. J Oral Pathol Med 1994;23:145–8.[CrossRef][Web of Science][Medline]
10 Ashby J, Styles JA, Boyland E. Betel nut, arecaidine, and oral cancer. Lancet 1979;1:112.[Web of Science][Medline]
11 Kuttan R, Donnely PV, Di Ferante N. Collagen treated with (+) catechin becomes resistant to the action of mammalian collagenase. Experentia 1981;37:221–3.[CrossRef][Web of Science][Medline]
12 Nishikawa A, Prokopczyk B, Rivenson A, Zang E, Hoffmann D. A study of betel quid carcinogenesis—VIII. Carcinogenicity of 3-(methylnitrosamino)propionaldehyde in F344 rats. Carcinogenesis 1992;13:369–72.
13 Meghji S, Scutt A, Harvey W, Canniff JP. An in-vitro comparison of human fibroblasts from normal and oral submucous fibrosis tissue. Arch Oral Biol 1987;32:213–5.[CrossRef][Web of Science][Medline]
14 Lee CH, Lin RH, Liu SH, Lin-Shiau SY. Mutual interactions among ingredients of betel quid in inducing genotoxicity on Chinese hamster ovary cells. Mutat Res 1996;367:99–104.[CrossRef][Web of Science][Medline]
15 Jeng JH, Kuo ML, Hahn LJ, Kuo MY. Genotoxic and non-genotoxic effects of betel quid ingredients on oral mucosal fibroblasts in vitro. J Dent Res 1994;73:1043–9.
16 Jeng JH, Lan WH, Hahn LJ, Hsieh CC, Kuo MY. Inhibition of the migration, attachment, spreading, growth and collagen synthesis of human gingival fibroblasts by arecoline, a major areca alkaloid, in vitro. J Oral Pathol Med 1996;25:371–5.[CrossRef][Web of Science][Medline]
17 Bauer HM, Ting Y, Greer CE, Chambers JC, Tashiro CJ, Chimera J, Reingold A, Manos MM. Genital human papillomavirus infection in female university students as determined by a PCR-based method. J Am Med Assoc 1991;265:472–7.
18 Lee YJ, Liao PH, Chen WK, Yang CC. Preferential cytotoxicity of caffeic acid phenethyl ester analogues on oral cancer cells. Cancer Lett 2000;153:51–6.[CrossRef][Web of Science][Medline]
19 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxity assays. J Immunol Methods 1983;65:55–63.[CrossRef][Web of Science][Medline]
20 Chen PC, Kuo C, Pan CC, Chou MY. Risk of oral cancer associated with human papilloma infection, betel quid chewing, and cigarette smoking in Taiwan—an integrated molecular and epidemiological study of 58 cases. J Oral Pathol Med 2002;31:317–22.[CrossRef][Web of Science][Medline]
21 Evans MF, Mount SL, Beatty BG, Cooper K. Biotinyl-tyramide-based in situ hybridization signal patterns distinguish human papillomavirus type and grade of cervical intraepithelial neoplasia. Mod Pathol 2002;15:1339–47.[CrossRef][Web of Science][Medline]
22 Laghi L, Randolph AE, Chauhan DP, Marra G, Major EO, Neel JV, Boland CR. JC virus DNA is present in the mucosa of the human colon and in colorectal cancers. Proc Natl Acad Sci USA 1999;96:7484–9.
23 Ko YC, Huang YL, Lee CH, Chen MJ, Lin LM, Tsai CC. Betel quid chewing, cigarette smoking and alcohol consumption related to oral cancer in Taiwan. J Oral Pathol Med 1995;24:450–3.[CrossRef][Web of Science][Medline]
24 Nair J, Ohshima H, Friesen M, Croisy A, Bhide SV, Bartsch H. Tobacco-specific and betel nut-specific N-nitrous compounds: occurrence in saliva and urine of betel quid chewers and formation in vitro by nitrosation of betel quid. Carcinogenesis 1985;6:295–303.
25 Franco EL. Cancer causes revisited: human papillomavirus and cervical neoplasia. J Natl Cancer Inst 1995;87:779–80.
26 Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummor JA, Shah KV, Snihders PJ, Peto J, Meijer CJ, Munoz N. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189:12–9.[CrossRef][Web of Science][Medline]
Received October 31, 2003; accepted March 1, 2004
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  N. Termine, V. Panzarella, S. Falaschini, A. Russo, D. Matranga, L. Lo Muzio, and G. Campisi HPV in oral squamous cell carcinoma vs head and neck squamous cell carcinoma biopsies: a meta-analysis (1988-2007) Ann. Onc., October 1, 2008; 19(10): 1681 - 1690. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||