EXPERIMENTAL CELL RESEARCH 226, 164–169 (1996) ARTICLE NO. 0215 HPV Immortalization of Human Oral Epithelial Cells: A Model for Carcinogenesis DOLPHINE ODA,* ,1 LENORA BIGLER,† PEGGY LEE,‡ AND REBECCA BLANTON‡ *Department of Oral Biology, University of Washington, Seattle, Washington 98195; †Department of Medicine, University of Mississippi Medical Center, University, Mississippi 38677; and ‡Fred Hutchinson Cancer Research Center, Seattle, Washington 98104 granular cell layers [1]. Although more than 70 dif- Human papillomavirus (HPV) has been implicated ferent types are now known [2], HPV shows a strong in the etiology of oral and cervical cancers. Normal type specificity for anatomical location and also for oral epithelial cells at passage two were infected characteristic type of lesions. Twenty-two types are with recombinant retrovirus containing the E6/E7 commonly found to infect the human anogenital tract open reading frames of HPV type 16. The G418-se- [3], and these virus types appear to have a role in lected cells that were immortalized and express HPV carcinogenesis of the anogenital region, in that more 16 E6/E7 have been in culture for over 4 years and than 90% of cervical cancers have been reported to 350 passages. In contrast, the normal oral epithelial contain HPV DNA [1]. HPV types 6 and 11 are found cells did not survive the culture environment beyond in benign condylomas, while types 16, 18, 31, and 32 7 to 9 passages. Fifteen clones were selected from are frequently associated with malignant progres- the pooled population. By Northern blot analysis all sion and cervical cancer [4–7]. clones demonstrated the presence of E6/E7 genes. One of the major problems in studying cellular Keratin expression of both normal and immortalized events during the development of human anogenital oral epithelial cells was studied in organotypic cul- ture. Both cell types were positive with antibodies cancer has been the lack of a suitable model system; AE1, AE3, and 34BE12. Both were focally positive however, this deficiency has been remedied by the with AE8, which stains for keratin 13 (specific for use of in vitro cell cultures which contain HPV. In- oral and esophageal epithelial cells). The normal sertion of HPV types 16 and 18 DNA in primary hu- control cells were focally positive for filaggrin, while man cervical epithelial [8] and foreskin epithelial the HPV 16-immortalized cells (IHGK cells—immor- cells [9] has resulted in immortalization of these talized human gingival keratinocytes) were nega- cells. DNA from type 6b, however, did not have im- tive. The IHGK cells were strongly positive with mortalizing capabilities [1, 9–13]. The ability to im- KS19.1, which stains the embryonal keratin K19, an mortalize has been localized to the E6/E7 open read- indicator of premalignant or malignant changes, ing frames and expression of these genes has been while the normal control cells were only lightly and demonstrated in cell lines immortalized with types focally positive. In conclusion, we present an oral 16 and 18 in vitro [12, 14–17]. epithelial cell line successfully immortalized with HPV E6/E7 which will facilitate further research on HPV also has a critical role in the etiology of oral the involvement of HPV in oral carcinogenesis. lesions. Twelve types of HPV are most commonly  1996 Academic Press, Inc. found in the oral cavity. Types 6, 11, 13, 16, and 32 are found in benign oral proliferative lesions such as condyloma acuminata and papillomas [18–20]. Types 2, 11, 16, 18, and 30 have been identified in INTRODUCTION oral epithelial dysplasias and squamous cell carcino- mas [21–26]. Human papilloma virus (HPV) is a DNA virus con- We have developed a practical and well-docu- sisting of 7900 bp that infects the basal epithelial mented model to facilitate the study of oral carcino- cells of skin and mucosal surfaces and replicates dur- genesis, through immortalization of normal human ing keratinocyte differentiation in the spinous and oral gingival keratinocytes with HPV 16 DNA [27, 28]. This process will allow the investigation of the 1 To whom correspondence and reprint requests should be ad- development of transformation in oral epithelial dressed at Division of Oral Pathology, Department of Oral Biology, cells and in particular the study of interactions of Box 357132, University of Washington, Seattle, WA 98195. Fax: (206) 685-3162; e-mail: doda@u.washington.edu. HPV with oral carcinogens such as alcohol and to- 164 0014-4827/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved. AID ECR 3225 / 6i11$$$$41 06-11-96 16:03:43 eca AP: Exp Cell