- Email: [email protected]
4NQO oral carcinogenesis: animal models, molecular markers and future expectations
Oral Oncology (2005) 41, 337–339
http://intl.elsevierhealth.com/journals/oron/
EDITORIAL REVIEW
4NQO oral carcinogenesis: animal models, molecular markers and future expectations Cancers of the oral cavity and oropharynx represent the sixth most common malignant neoplasms with an estimated annual worldwide incidence of 500,000 cases.1 The American Cancer Society estimates that approximately 30,000 new cases of, oral cancer were diagnosed in 2002 and nearly 7,400 people will die from this disease.2 Squamous cell carcinoma (SCC), which arises from the oral mucosa lining, accounts for over 90% of these tumors.3 It has been postulated that tumorigenesis is a multistep process and requires accumulation of carcinogen-induced genetic changes throughout the exposed tissue field. The concept of field cancerization, established in 1953, is based on the fact that the epithelial surface of the upper airway can be exposed to the same common carcinogens. Thus, it possesses an increased risk in the development of oral cancer development.4 The animal model of 4NQO carcinogenesis, by systemic administration in drinking water or by topical application, produces a spectrum of preneoplastic and neoplastic lesions and, thus is considered to be the preferred model to study oral squamous cell carcinoma (OSCC).5 Its advantage lies in the fact that it parallels the development of OSCC in humans, i.e., the dysplastic lesions are produced by long-term ingestion of small amounts of carcinogen.5 Since the original 4NQO carcinogenesis model of Wallenius and Leckholm,6 where repeated applications of the water-soluble carcinogen to the rat palate induced epithelial dysplasia and ultimately SCC, a series of studies published in the English language literature have provided abundant evidence for a well-established relationship between histologic features of epithelial dysplasia and time of application of the carcinogen.5,7–13 Although the experimental protocols used in these studies vary
widely in relation to duration of carcinogen application and sacrifice time points, it was generally found that exposing the rat palatal mucosa to 4NQO carcinogen produces initial disturbances in the morphology of the palatal epithelium compatible with dysplasia. In one study it was shown that this exposure can be as short as 2 weeks.10 In other studies, the duration of carcinogen application required for the appearance of the first dysplastic changes was reported as usually longer: 4,9,12,13 88,11,13 and 12 weeks.7 It is important to note that studies, which concomitantly analyzed changes in the histological features and clinical appearance of the palatal mucosa, reported a latent period between the two. The first histological changes usually preceded clinical modifications8,10,13 for as long as 8 weeks.8,13 A similar pattern of changes with a latency period was reported when 4NQO carcinogen was administered in drinking water and rat tongues were analyzed for clinical and histological alterations.5 Clinical and histological evidence that the rat 4NQO model of oral carcinogenesis simulates the development of human SCC within premalignant white oral lesions was only the first step. From the beginning of this model, it was felt that both the gross clinical and fine microscopic findings are rather late morphologic changes and earlier substantial events at the nuclear and molecular level precede their appearance. Thus, several markers with an alleged potential to indicate and predict the evolvement of a truly invasive malignant neoplasm following exposure to 4NQO have been investigated for some time. This included markers, such as nuclear profile of basal epithelial cells,9 cytokeratin profile,14 AgNOR parameters,15,16 proliferating markers PCNA17 and BrdU,18 tumor suppressive protein p5319 and cadherin-associated proteins.20 It was generally found that changes in
1368-8375/$ - see front matter c 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2004.07.005
338 these markers became more intense as the oral epithelium progressed histologically from normal to dysplastic to carcinoma. In the particular case of the AgNOR method, intranuclear changes, comprised of a significant increase in number and area of AgNORs, were identified much earlier (14 weeks) than any clinical modifications could be observed.16 In addition, this model has been investigated at a genetic level to trace the earliest key changes in charge of the malignant transformation. For example, high frequency of loss of heterozygosity (LOH) in loci harboring oncogenes (e.g., Ha-ras) has been found.21 Studies, in vivo and in vitro, have shown that as 4NQO-induced carcinogenesis progresses, the frequency of changes in DNA content, (i.e., ploidy), increases, usually featuring the appearance of hyperploid cells.18,22,23 Whether this event precedes the acquirement of a malignant phenotype (i.e., anchorage independence and tumorogenicity in athymic mice) remains a matter of debate.23 In the present issue of Oral Oncology, Ribeiro et al. confirms and further supports the idea that 4NQO-induced DNA damage is an early event and that it can be present in what appears to be both clinically and histologically normal epithelium. A relatively uncommon diagnostic tool in the study of oral cancer, the single cell gel (comet) assay, is also introduced.24 The above reviewed markers and diagnostic methods as well as their results have served as a valuable source for investigating human oral carcinogesis. We must now find reliable markers to signal and predict the risk of transition of oral premalignant lesions (leukoplakias) to a malignant stage. However, each time a new marker is suggested, it actually poses a new hurdle. It should be remembered the enthusiasm over the tumor suppressor protein p53. In time, it was concluded that either gene mutation or change in protein expression could be associated with cellular response to environmental stimuli and not necessarily with malignant transformation.25,26 The AgNOR method yielded contradictory results, thus its predictive value is still uncertain.27,28 LOH at specific loci was found in increasing frequencies as oral premalignant lesions showed histological advance toward true malignancy. However, it is not known as yet, what risks are associated with other identifiable LOH.29 Another marker recently proposed as a predictive factor for malignant transformation of oral leukoplakias is the measurement of DNA content.30 According to the study, dysplastic leukoplakias found in diploid lesions are considered as low risk for malignant transformation, while aneuploid lesions are considered as high risk. In addition, the degree of the dysplasia found in these
Editorial Review leukoplakias did not correlate with DNA content or the risk of cancer. Although extremely promising, before this marker can be clinically applicable, clarification is needed as to what occurs in the oral cancers that do not develop at a previous leukoplakia site. This is true for about half of the cases.31 Furthermore, aneuploidy cannot be used as a predictive marker in diploid oral cancers, which, again, constitute about half of all cases.31 Thus far, the most predictive markers assessed for oral SCC include cancer history, the presence of epithelial dysplasia, chromosomal aneuploidy, p53 expression, and LOH at chromosome 3p or 9p.32 The stronger predictors for malignancy were found to be histology and the combined biomarker score of chromosomal polysomy, p53 and LOH.33 Use of these markers as an adjunctive to routine histopathologic examination is necessary to achieve the most accurate prognostic potential and, subsequently, the most effective management of the premalignant lesions.32,33 What becomes clear from these findings and subsequent asked questions, is that neither a single molecular marker/class of markers nor one investigative method is powerful enough to predict the risk of oral cancer development in each individual. Future investigations are mandatory that would validate the above predictive molecular markers in cells collected from either oral mucosa smears or whole saliva in high-risk patients with no visible clinical changes of the oral mucosa. The animal model of 4NQO oral carcinogenesis can be helpful in conducting preliminary studies on genomic instability of epithelial cells collected either from oral smears or from whole saliva. Meanwhile, on more practical grounds, the American Cancer Society recommends an annual check-up for all individuals over the age of 40, and every 3 years for individuals between ages of 20 and 39 years, which ‘‘should include health counseling and, depending on a personÕs age, might include examinations for cancers of the thyroid, oral cavity, skin, lymph nodes, testes and ovaries’’.34
References 1. Pisani B, Bray F, Parkin DM. Estimates of the world-wide prevalence of cancer for 25 sites in the adult population. Int J Cancer 2002;97:72–81. 2. Neville BW, Day TA. Oral cancer and precancerous lesions. CA Cancer J Clin 2002;52:195–215. 3. Silverman Jr S. Demographics and occurrence of oral and oropharyngeal cancers. The outcomes, the trends, the challenge. J Am Dent Assoc 2001;132:75–115. 4. Slaughter DP, Southwick HW, Smejkal W. Field cancerization in oral stratified squamous epithelium: clinical implications of multicenter origin. Cancer (Phila) 1953;6:963–8.
Editorial Review 5. Dayan D, Hirshberg A, Kaplan I, Rotem N, Bodner L. Experimental tongue cancer in desalivated rats. Oral Oncol 1997;33:105–9. 6. Wallenius K, Lekholm U. Oral cancer in rats induced by the water soluble carcinogen 4-nitroquinoline 1-oxide. Odontol Revy 1973;24:39–48. 7. Prime SS, Malamos D, Rosser T, Scully C. Oral epithelial atypia and acantholytic dyskeratosis in rats painted with 4nitroquinoline N-oxide. J Oral Pathol 1986;15:280–3. 8. Dayan D, Kaplan I, Russak S, Bodner L. Dysplastic changes of the palate and tongue mucosa induced by topically applied 4 nitroquinoline-N-oxide (4NQO) in desalivated rats. In: Varma AK, editor. Proceedings of the 5th international congress on oral cancer, Oral oncology, vol. 5. New Delhi: MacMillan India; 1997. p. 514–7. 9. Rich AM, Reade PC. Nuclear morphometry in experimental oral mucosal carcinogenesis. Eur J Cancer B Oral Oncol 1996;32B:169–75. 10. Nauta JM, Roodenburg JLN, Nikkels PGJ, Witjes MJH, Vermey A. Epithelial dysplasia and squamous cell carcinoma of the Wistar rat palatal mucosa: 4NQO model. Head Neck 1996;18:441–9. 11. Wong PNC, Wilson DF. 4 Nitroquinoline 1-oxide-induced carcinogenesis in the rat palate. J Oral Pathol 1983;12:375–84. 12. Kandakar SV, Sawant SS, Reade PC. Ultrastructural changes to the palatal mucosa of rats following the application of 4nitroquinoline-l-oxide (4NQO) and vitamin C. Oral Oncol 1998;34:247–52. 13. Rich AM, Reade PC. Histomorphometric analysis of epithelial changes in chemically induced oral mucosal carcinogenesis in rats. J Oral Pathol 1988;17:528–33. 14. Boyd NM, Reade PC. Temporal alterations in cytokeratin expression during experimental oral mucosal carcinogenesis. Carcinogenesis 1991;12:1767–71. 15. Tanaka T, Kojima T, Okumura A, Yoshimi N, Mori H. Alterations of the nucleolar organizer regions during 4nitroquinoline 1-oxide-induced tongue carcinogenesis. Carcinogenesis 1991;12:329–33. 16. Kaplan I, Engelberg J, Dayan D. The effect of desalivation on 4- nitroquinoline-1-oxide-induced tongue carcinogenesis: a morphometric study of nucleolar organizer regions. J Oral Pathol Med 2001;30:48–52. 17. Kaplan I, Hochstadt T, Dayan D. PCNA in palate and tongue mucosal dysplastic lesions induced by topically applied 4NQO in desalivated rat. Med Oral 2002;7:336–43. 18. Itoh T, Yamamoto Y, Saka T, Inoue I, Takahashi H. Estimation of proliferative activity of experimental tongue carcinoma in rats. Immunohistochemical and DNA cytofluorometric analysis. Acta Otolaryngol 1993;113: 568–74. 19. Osugi Y. p53 expression in various stages of 4-nitroquinoline 1-oxide induced carcinoma in rat tongue. J Osaka Dent Univ 1996;30:29–35. 20. Sakaki T, Tamura I, Katoda H, Kakudo K. Changing expression of E- and P-cadherin during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide. J Oral Pathol Med 2003;32:530–7. 21. Tanuma J, Hiai H, Shisa H et al. Carcinogenesis modifier loci in rat tongue are subject to frequent loss of heterozygosity. Int J Cancer 2002;102:638–42.
339 22. Wallenius K, Ahlstrom U, Heyden G. Cytogenetic and histochemical analysis of experimental oral carcinogenesis in the rat. Odontol Revy 1975;26:309–26. 23. Crane IJ, Patel V, Scully C, Prime SS. Development of aneuploidy in experimental oral carcinogenesis. Carcinogenesis 1989;10:2375–7. 24. Tice RR, Agurell E, Anderson D et al. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 2000;35:206–21. 25. Kaplan I, Vered M, Moskona D, Buchner A, Dayan D. An immunohistochemical study of p53 and PCNA in inflammatory papillary hyperplasia of the palate: a dilemma of interpretation. Oral Dis 1998;4:194–9. 26. Zhang Z, Suo Z, Sudbo J, Holm R, Boysen M, Reith A. Diagnostic implications of p53 protein reactivity in nasal mucosa of nickel workers. Anal Quant Cytol Histol 1997;19:345–50. 27. Chattopadhyah A, Ray JG, Caplan DJ. AgNOR count as objective marker for dysplastic features in oral leukoplakia. J Oral Pathol Med 2002;31:512–7. 28. Kahn MA, Mincer HH, Dockter ME, Hermann-Petrin JM. Comparing flow cytometric analysis and nucleolar organizer region enumeration in archival oral premalignant lesions. J Oral Pathol Med 1993;22:257–62. 29. Reith A, Sudbo J. Impact of genomic instability in risk assessment and chemoprevention of oral premalignancies. Int J Cancer 2002;101:205–9. 30. Sudbo J, Kildal W, Risberg B, Koppang HS, Danielsen HE, Reith A. DNA content as a prognostic marker in patients with oral leukoplakia. N Engl J Med 2001;344:1270–8. 31. Lipmann SM, Hong WK. Molecular markers of the risk of oral cancer [editorial]. N Engl J Med 2001;344:1323–6. 32. Scully C, Sudbo J, Speight PM. Progress in determining the malignant potential of oral lesions. Review article. J Oral Pathol Med 2003;32:251–6. 33. Tsao AS, Kim ES, Hong WK. Chemoprevention of cancer. CA Cancer J Clin 2004;54:150–80. 34. United States Preventive Services Task Force (USPSTF). In: Guide to clinical preventive services: report of the United States Preventive Services Task Force. 2nd ed. Baltimore Williams & Wilkins.; 1996 [accessed on May 2002].
Marilena Vered Noam Yarom Dan Dayan Department of Oral Pathology and Oral Medicine The Maurice and Gabriela Goldschleger School of Dental Medicine Tel Aviv University Tel Aviv, Israel Tel.: +972 3 640 9305/9112; fax: +972 3 640 9250 E-mail address: [email protected] (D. Dayan). Received 27 July 2004; accepted 28 July 2004
http://intl.elsevierhealth.com/journals/oron/
EDITORIAL REVIEW
4NQO oral carcinogenesis: animal models, molecular markers and future expectations Cancers of the oral cavity and oropharynx represent the sixth most common malignant neoplasms with an estimated annual worldwide incidence of 500,000 cases.1 The American Cancer Society estimates that approximately 30,000 new cases of, oral cancer were diagnosed in 2002 and nearly 7,400 people will die from this disease.2 Squamous cell carcinoma (SCC), which arises from the oral mucosa lining, accounts for over 90% of these tumors.3 It has been postulated that tumorigenesis is a multistep process and requires accumulation of carcinogen-induced genetic changes throughout the exposed tissue field. The concept of field cancerization, established in 1953, is based on the fact that the epithelial surface of the upper airway can be exposed to the same common carcinogens. Thus, it possesses an increased risk in the development of oral cancer development.4 The animal model of 4NQO carcinogenesis, by systemic administration in drinking water or by topical application, produces a spectrum of preneoplastic and neoplastic lesions and, thus is considered to be the preferred model to study oral squamous cell carcinoma (OSCC).5 Its advantage lies in the fact that it parallels the development of OSCC in humans, i.e., the dysplastic lesions are produced by long-term ingestion of small amounts of carcinogen.5 Since the original 4NQO carcinogenesis model of Wallenius and Leckholm,6 where repeated applications of the water-soluble carcinogen to the rat palate induced epithelial dysplasia and ultimately SCC, a series of studies published in the English language literature have provided abundant evidence for a well-established relationship between histologic features of epithelial dysplasia and time of application of the carcinogen.5,7–13 Although the experimental protocols used in these studies vary
widely in relation to duration of carcinogen application and sacrifice time points, it was generally found that exposing the rat palatal mucosa to 4NQO carcinogen produces initial disturbances in the morphology of the palatal epithelium compatible with dysplasia. In one study it was shown that this exposure can be as short as 2 weeks.10 In other studies, the duration of carcinogen application required for the appearance of the first dysplastic changes was reported as usually longer: 4,9,12,13 88,11,13 and 12 weeks.7 It is important to note that studies, which concomitantly analyzed changes in the histological features and clinical appearance of the palatal mucosa, reported a latent period between the two. The first histological changes usually preceded clinical modifications8,10,13 for as long as 8 weeks.8,13 A similar pattern of changes with a latency period was reported when 4NQO carcinogen was administered in drinking water and rat tongues were analyzed for clinical and histological alterations.5 Clinical and histological evidence that the rat 4NQO model of oral carcinogenesis simulates the development of human SCC within premalignant white oral lesions was only the first step. From the beginning of this model, it was felt that both the gross clinical and fine microscopic findings are rather late morphologic changes and earlier substantial events at the nuclear and molecular level precede their appearance. Thus, several markers with an alleged potential to indicate and predict the evolvement of a truly invasive malignant neoplasm following exposure to 4NQO have been investigated for some time. This included markers, such as nuclear profile of basal epithelial cells,9 cytokeratin profile,14 AgNOR parameters,15,16 proliferating markers PCNA17 and BrdU,18 tumor suppressive protein p5319 and cadherin-associated proteins.20 It was generally found that changes in
1368-8375/$ - see front matter c 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2004.07.005
338 these markers became more intense as the oral epithelium progressed histologically from normal to dysplastic to carcinoma. In the particular case of the AgNOR method, intranuclear changes, comprised of a significant increase in number and area of AgNORs, were identified much earlier (14 weeks) than any clinical modifications could be observed.16 In addition, this model has been investigated at a genetic level to trace the earliest key changes in charge of the malignant transformation. For example, high frequency of loss of heterozygosity (LOH) in loci harboring oncogenes (e.g., Ha-ras) has been found.21 Studies, in vivo and in vitro, have shown that as 4NQO-induced carcinogenesis progresses, the frequency of changes in DNA content, (i.e., ploidy), increases, usually featuring the appearance of hyperploid cells.18,22,23 Whether this event precedes the acquirement of a malignant phenotype (i.e., anchorage independence and tumorogenicity in athymic mice) remains a matter of debate.23 In the present issue of Oral Oncology, Ribeiro et al. confirms and further supports the idea that 4NQO-induced DNA damage is an early event and that it can be present in what appears to be both clinically and histologically normal epithelium. A relatively uncommon diagnostic tool in the study of oral cancer, the single cell gel (comet) assay, is also introduced.24 The above reviewed markers and diagnostic methods as well as their results have served as a valuable source for investigating human oral carcinogesis. We must now find reliable markers to signal and predict the risk of transition of oral premalignant lesions (leukoplakias) to a malignant stage. However, each time a new marker is suggested, it actually poses a new hurdle. It should be remembered the enthusiasm over the tumor suppressor protein p53. In time, it was concluded that either gene mutation or change in protein expression could be associated with cellular response to environmental stimuli and not necessarily with malignant transformation.25,26 The AgNOR method yielded contradictory results, thus its predictive value is still uncertain.27,28 LOH at specific loci was found in increasing frequencies as oral premalignant lesions showed histological advance toward true malignancy. However, it is not known as yet, what risks are associated with other identifiable LOH.29 Another marker recently proposed as a predictive factor for malignant transformation of oral leukoplakias is the measurement of DNA content.30 According to the study, dysplastic leukoplakias found in diploid lesions are considered as low risk for malignant transformation, while aneuploid lesions are considered as high risk. In addition, the degree of the dysplasia found in these
Editorial Review leukoplakias did not correlate with DNA content or the risk of cancer. Although extremely promising, before this marker can be clinically applicable, clarification is needed as to what occurs in the oral cancers that do not develop at a previous leukoplakia site. This is true for about half of the cases.31 Furthermore, aneuploidy cannot be used as a predictive marker in diploid oral cancers, which, again, constitute about half of all cases.31 Thus far, the most predictive markers assessed for oral SCC include cancer history, the presence of epithelial dysplasia, chromosomal aneuploidy, p53 expression, and LOH at chromosome 3p or 9p.32 The stronger predictors for malignancy were found to be histology and the combined biomarker score of chromosomal polysomy, p53 and LOH.33 Use of these markers as an adjunctive to routine histopathologic examination is necessary to achieve the most accurate prognostic potential and, subsequently, the most effective management of the premalignant lesions.32,33 What becomes clear from these findings and subsequent asked questions, is that neither a single molecular marker/class of markers nor one investigative method is powerful enough to predict the risk of oral cancer development in each individual. Future investigations are mandatory that would validate the above predictive molecular markers in cells collected from either oral mucosa smears or whole saliva in high-risk patients with no visible clinical changes of the oral mucosa. The animal model of 4NQO oral carcinogenesis can be helpful in conducting preliminary studies on genomic instability of epithelial cells collected either from oral smears or from whole saliva. Meanwhile, on more practical grounds, the American Cancer Society recommends an annual check-up for all individuals over the age of 40, and every 3 years for individuals between ages of 20 and 39 years, which ‘‘should include health counseling and, depending on a personÕs age, might include examinations for cancers of the thyroid, oral cavity, skin, lymph nodes, testes and ovaries’’.34
References 1. Pisani B, Bray F, Parkin DM. Estimates of the world-wide prevalence of cancer for 25 sites in the adult population. Int J Cancer 2002;97:72–81. 2. Neville BW, Day TA. Oral cancer and precancerous lesions. CA Cancer J Clin 2002;52:195–215. 3. Silverman Jr S. Demographics and occurrence of oral and oropharyngeal cancers. The outcomes, the trends, the challenge. J Am Dent Assoc 2001;132:75–115. 4. Slaughter DP, Southwick HW, Smejkal W. Field cancerization in oral stratified squamous epithelium: clinical implications of multicenter origin. Cancer (Phila) 1953;6:963–8.
Editorial Review 5. Dayan D, Hirshberg A, Kaplan I, Rotem N, Bodner L. Experimental tongue cancer in desalivated rats. Oral Oncol 1997;33:105–9. 6. Wallenius K, Lekholm U. Oral cancer in rats induced by the water soluble carcinogen 4-nitroquinoline 1-oxide. Odontol Revy 1973;24:39–48. 7. Prime SS, Malamos D, Rosser T, Scully C. Oral epithelial atypia and acantholytic dyskeratosis in rats painted with 4nitroquinoline N-oxide. J Oral Pathol 1986;15:280–3. 8. Dayan D, Kaplan I, Russak S, Bodner L. Dysplastic changes of the palate and tongue mucosa induced by topically applied 4 nitroquinoline-N-oxide (4NQO) in desalivated rats. In: Varma AK, editor. Proceedings of the 5th international congress on oral cancer, Oral oncology, vol. 5. New Delhi: MacMillan India; 1997. p. 514–7. 9. Rich AM, Reade PC. Nuclear morphometry in experimental oral mucosal carcinogenesis. Eur J Cancer B Oral Oncol 1996;32B:169–75. 10. Nauta JM, Roodenburg JLN, Nikkels PGJ, Witjes MJH, Vermey A. Epithelial dysplasia and squamous cell carcinoma of the Wistar rat palatal mucosa: 4NQO model. Head Neck 1996;18:441–9. 11. Wong PNC, Wilson DF. 4 Nitroquinoline 1-oxide-induced carcinogenesis in the rat palate. J Oral Pathol 1983;12:375–84. 12. Kandakar SV, Sawant SS, Reade PC. Ultrastructural changes to the palatal mucosa of rats following the application of 4nitroquinoline-l-oxide (4NQO) and vitamin C. Oral Oncol 1998;34:247–52. 13. Rich AM, Reade PC. Histomorphometric analysis of epithelial changes in chemically induced oral mucosal carcinogenesis in rats. J Oral Pathol 1988;17:528–33. 14. Boyd NM, Reade PC. Temporal alterations in cytokeratin expression during experimental oral mucosal carcinogenesis. Carcinogenesis 1991;12:1767–71. 15. Tanaka T, Kojima T, Okumura A, Yoshimi N, Mori H. Alterations of the nucleolar organizer regions during 4nitroquinoline 1-oxide-induced tongue carcinogenesis. Carcinogenesis 1991;12:329–33. 16. Kaplan I, Engelberg J, Dayan D. The effect of desalivation on 4- nitroquinoline-1-oxide-induced tongue carcinogenesis: a morphometric study of nucleolar organizer regions. J Oral Pathol Med 2001;30:48–52. 17. Kaplan I, Hochstadt T, Dayan D. PCNA in palate and tongue mucosal dysplastic lesions induced by topically applied 4NQO in desalivated rat. Med Oral 2002;7:336–43. 18. Itoh T, Yamamoto Y, Saka T, Inoue I, Takahashi H. Estimation of proliferative activity of experimental tongue carcinoma in rats. Immunohistochemical and DNA cytofluorometric analysis. Acta Otolaryngol 1993;113: 568–74. 19. Osugi Y. p53 expression in various stages of 4-nitroquinoline 1-oxide induced carcinoma in rat tongue. J Osaka Dent Univ 1996;30:29–35. 20. Sakaki T, Tamura I, Katoda H, Kakudo K. Changing expression of E- and P-cadherin during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide. J Oral Pathol Med 2003;32:530–7. 21. Tanuma J, Hiai H, Shisa H et al. Carcinogenesis modifier loci in rat tongue are subject to frequent loss of heterozygosity. Int J Cancer 2002;102:638–42.
339 22. Wallenius K, Ahlstrom U, Heyden G. Cytogenetic and histochemical analysis of experimental oral carcinogenesis in the rat. Odontol Revy 1975;26:309–26. 23. Crane IJ, Patel V, Scully C, Prime SS. Development of aneuploidy in experimental oral carcinogenesis. Carcinogenesis 1989;10:2375–7. 24. Tice RR, Agurell E, Anderson D et al. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 2000;35:206–21. 25. Kaplan I, Vered M, Moskona D, Buchner A, Dayan D. An immunohistochemical study of p53 and PCNA in inflammatory papillary hyperplasia of the palate: a dilemma of interpretation. Oral Dis 1998;4:194–9. 26. Zhang Z, Suo Z, Sudbo J, Holm R, Boysen M, Reith A. Diagnostic implications of p53 protein reactivity in nasal mucosa of nickel workers. Anal Quant Cytol Histol 1997;19:345–50. 27. Chattopadhyah A, Ray JG, Caplan DJ. AgNOR count as objective marker for dysplastic features in oral leukoplakia. J Oral Pathol Med 2002;31:512–7. 28. Kahn MA, Mincer HH, Dockter ME, Hermann-Petrin JM. Comparing flow cytometric analysis and nucleolar organizer region enumeration in archival oral premalignant lesions. J Oral Pathol Med 1993;22:257–62. 29. Reith A, Sudbo J. Impact of genomic instability in risk assessment and chemoprevention of oral premalignancies. Int J Cancer 2002;101:205–9. 30. Sudbo J, Kildal W, Risberg B, Koppang HS, Danielsen HE, Reith A. DNA content as a prognostic marker in patients with oral leukoplakia. N Engl J Med 2001;344:1270–8. 31. Lipmann SM, Hong WK. Molecular markers of the risk of oral cancer [editorial]. N Engl J Med 2001;344:1323–6. 32. Scully C, Sudbo J, Speight PM. Progress in determining the malignant potential of oral lesions. Review article. J Oral Pathol Med 2003;32:251–6. 33. Tsao AS, Kim ES, Hong WK. Chemoprevention of cancer. CA Cancer J Clin 2004;54:150–80. 34. United States Preventive Services Task Force (USPSTF). In: Guide to clinical preventive services: report of the United States Preventive Services Task Force. 2nd ed. Baltimore Williams & Wilkins.
Marilena Vered Noam Yarom Dan Dayan Department of Oral Pathology and Oral Medicine The Maurice and Gabriela Goldschleger School of Dental Medicine Tel Aviv University Tel Aviv, Israel Tel.: +972 3 640 9305/9112; fax: +972 3 640 9250 E-mail address: [email protected] (D. Dayan). Received 27 July 2004; accepted 28 July 2004