[CANCER RESEARCH 52. 4254-4260, August I, 1992] Squamous Metaplasia of Normal and Carcinoma in Situ of HPV 16-Immortalized Human Endocervical Cells1 Qi Sun, Kouichiro Tsutsumi, M. Brian Kelleher, Alan Pater, and Mary M. Pater2 Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada A1B ÃŒV6 ABSTRACT genomic DNA, most frequently of HPV 16, has been detected in 90% of the cervical carcinomas and are found to be actively The importance of cervical squamous metaplasia and human papil- expressed (6, 7). HPV 16 DNA has been used to transform lomavirus 16 (HPV 16) infection for cervical carcinoma has been well human foreskin and ectocervical keratinocytes (8, 9). It immor established. Nearly 87% of the intraepithelial neoplasia of the cervix occur in the transformation zone, which is composed of squamous meta- talizes human keratinocytes efficiently, producing cell clones plastic cells with unclear origin. HPV DNA, mostly HPV 16, has been with indefinite life span in culture. Different approaches have found in 90% of cervical carcinomas, but only limited experimental data been taken to examine the behavior of these immortalized cell are available to discern the role of HPV 16 in this tissue specific onco- lines in conditions allowing squamous differentiation (10, 11). genesis. We have initiated in vivo studies of cultured endocervical cells After transplantation in vivo, the HPV 16-immortalized kerat as an experimental model system for development of cervical neoplasia. inocytes retain thépotential for squamous differentiation, Using a modified in vivo implantation system, cultured normal endocer forming abnormal epithelium without dysplastic changes at vical epithelial cells formed epithelium resembling squamous metapla early passages and with various dysplastic changes only after sia, whereas those immortalized by HPV 16 developed into lesions long periods of time in culture (10). resembling carcinoma in situ. In contrast, their ectocervical counter Considering that most cases of cervical neoplasia are located parts formed well differentiated stratified squamous epithelium and a lesion with mild dysplastic change, respectively. The HPV 16-immor- within the transformation zone and the endocervix (3), it ap talized cells showed in vivo cytokeratin expression patterns similar to peared logical that cultured endocervical cells should be the their respective normal counterparts, confirming their different origins. most appropriate system to study cervical oncogenesis by HPV. Thus, this study provides direct experimental evidence for the transfor Previously we found that HPV 16 DNA can immortalize hu mation of simple epithelial cells of endocervical origin into stratified man endocervical cells in vitro (12). We report here that, upon squamous metaplasia and indicates the differential susceptibility of en- being implanted into athymic mice, the normal endocervical do- and ectocervical epithelial cells for conversion to cancer by HPV 16. cells formed stratified squamous epithelium resembling squa mous metaplasia, and HPV 16-immortalized endocervical cell INTRODUCTION implants displayed a pathological picture analogous to that of carcinoma in situ. Squamous cell carcinoma of the uterine cervix is one of the most common neoplasms in the world (1). The normal cervix is comprised of 2 distinct regions, the ectocervix and endocervix MATERIALS AND METHODS (2). The former is lined with stratified, nonkeratinizing squa Cell Culture. Endo- and ectocervical epithelial cell cultures were mous epithelium (native squamous epithelium) and the latter derived from cervical specimens obtained from hysterectomies per with simple columnar epithelium. The junction where the 2 formed for benign conditions. Primary cultures of endo- and ectocer regions adjoin, the squamo-columnar junction, undergoes dy vical epithelial cells were initiated and maintained as described (12, 13) namic modification. Under certain physiological or pathologi from cervical regions distant from squamo-columnar junctions. All tis cal conditions, the simple epithelium at the squamo-columnar sue culture was performed with KGM (Clonetics, San Diego, CA). Cells junction is replaced by stratified squamous epithelium (or meta- were passaged 1:2 when 60-80% confluent for primary cultures and 1:3 plastic squamous epithelium), giving rise to a region referred to when 90% confluent for immortalized cells. as the transformation zone (2). The transformation zone is the Transmission Electron Microscopy. Trypsinized cells were seeded most common site for development of cervical neoplasia (3). into Millicell-HA cups (Millipore, Bedford, MA). Upon confluence, the cells together with the cellulose-ester membranes were fixed in 5% The origin of cervical squamous metaplastic cells has been glutaraldehyde and postfixed with 4% OsO.,. Epon 812-embedded thin hypothesized to be either endocervical epithelial cells (4) or sections were stained with uranyl acetate and lead citrate. Thin sections subepithelial stromal cells (5). It has been well established that certain types of HPVs3 are were examined with a Jeol 1200 electron microscope. Cell Implantation in Nude Mice and Preparation for Histology. involved in the oncogenesis of cervical carcinomas. HPV Trypsinized cells (IO6 to IO7) were seeded into 35-mm Petri dishes in KGM, in the center of which a 1.5 x 1.5-cm sterile silicone sheet (Dow Received 2/21/92; accepted 5/12/92. Corning, Midland, MI) had been placed. The medium was changed The costs of publication of this article were defrayed in part by the payment of after 8 h, and after 48 h the silicone sheet with attached cells was page charges. This article must therefore be hereby marked advertisement in accord implanted into 2-3-month-old female nude mice (nu/nu; NIH) as de ance with 18 U.S.C. Section 1734 solely to indicate this fact. 1The investigation was supported in part by grants awarded by the National scribed by Barrandon and Green (14). The implants together with the Cancer Institute of Canada (with funds from the Canadian Cancer Society) and the skin flaps were recovered after 8 days, fixed with 4% paraformaldehyde Medical Research Council of Canada to A. P. and M. M. P. in phosphate buffered saline, embedded in paraffin, sectioned, and 2 To whom requests for reprints should be addressed, at Memorial University of stained with hematoxylin and eosin. Newfoundland, Faculty of Medicine, Division of Basic Medical Sciences, St. John's, Newfoundland, Canada AI B 3V6. Mucin and Cytokeratin Staining. For detecting mucin, unstained 3 The abbreviations used are: HPV, human papillomavirus; CK, cytokeratin; sections prepared for histology were stained with Alcian blue at pH 2.5 CIN, cervical intraepithelial neoplasia; HEN, cultured human endocervical epithe (15). lial cells; HEC, cultured human ectocervical epithelial cells; HEN-16, cell line from cultured human endocervical epithelial cells immortalized by HPV 16; HEC-16, For cytokeratin staining (16), the unstained sections prepared for histology were deparaffined and rehydrated. They were treated with cell line from cultured human ectocervical epithelial cells immortalized by HPV 16; 0.25% trypsin (Sigma, St. Louis, MO) at 37°Cfor30 min for antibodies KGM, keratinocyte growth medium. 4254 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1992 American Association for Cancer Research. IN VIVO IMPLANTS OF HUMAN ENDOCERVICAL CELLS from Sigma or 2 h for antibody from ICN (ICN Biomedicals, Lisle, IL) and then incubated at 4°Covernight with the mouse anti-cytokeratin monoclonal antibodies to CK18 (CY-90), CK13 (Ks-lA3) and CK10 (K8.60) (Sigma), and CK19 (Ks.19.1) (ICN) with dilutions recom mended by the suppliers. The sections were stained with fluorescein isothiocyanate-labeled goat anti-mouse IgG (Sigma) for l h at room temperature. Observation was made with a Leitz Diaplan microscope. RNA Preparation and Northern Blot Analysis. Total cellular RNA was prepared from cells in culture and analyzed by Northern blot hy bridization as described previously (17). The probes were labeled with -12Pusing a random priming kit (BRL, Gaithersburg, MD). All washing was in 0.1 x sodium saline citrate and 0.1% sodium dodecyl sulfate at 65°C. RESULTS HEN-16 and HEC-16 are immortalized cell lines that were derived from normal human endo- and ectocervical epithelial cells, respectively, transfected with whole genomic HPV 16 DNA. They both contain integrated HPV 16 genomes and ex press viral messages (12). In Vitro Ultrastructure of HPV 16-immortalized Endocervi- cal Cells. Primary cultures of endocervical cells display pleo- morphism in culture, which evolves with passage (13). HPV 16 DNA transfection was performed to derive HEN-16, using a primary parental endocervical cell culture that consisted of 2 cell populations morphologically discernible with the light mi croscope. Electron microscopy also showed that these endocer vical cells cultured in KGM have an ultrastructure different from those of ectocervical cells (13). To evaluate the effects of immortalization on endocervical cell morphology, cells were examined with electron micro scopy. For maintaining intercellular structure, the cells were Fig. 1. Ultrastructure of HPV to-immortalized endo- and ectocervical cells. grown and fixed in situ on membranes. HEN-16 showed mod Bar (a), 500 nm. Original magnifications x 18,000. D, desmosome; M, microvilli; erate amounts of tonofilament bundles, well developed desmo- T, tonofilament.