GYNECOLOGIC

ONCOLOGY

38, 407-412

(1990)

Human Cervical Cells Immortalized in Vitro with Oncogenic Human Papillomavirus DNA Differentiate Dysplastically in Viva S. E.

WAGGONER,*

C. D.

*Division Cancer

o.fGynrcokjgic Oncology, Georgefown University Medical Center, Wushington, DC, 20007; tLahoratory qf Pathology and Luhorutoty Medicine, University Institute. Bethesda. Maryland 20892: and $Depurtmrnt of Medicine und Dentistry, RochestcJr, Ne,t, York 14627

WOODWORTH,~

M. H. STOLER,~ W. A. BARNES,* G. DELGADO,”

Received

J. A. DIPAOLO~ of Biology, oj’Rochrster

National School

January 22. 1990

Human papillomavirus (HPV) types 16 and 18 are associated with cervical dysplasia and carcinoma. In vitro integration of HPV-16 or HPV-18 DNA into cultured human cervical cells results in their immortalization. In this study, in vivo differentiation of human cervical cells immortalized with recombinant HPV-16 or HPV-18 DNA was studied by implanting confluent monolayers of HPV-16 and HPV-18 DNA immortalized cell lines under dorsal skin flaps of immunodeficient mice. Grafts of a human cervical cancer cell line (C4-1) and cultured normal cervical cells served as controls. Histologic analysis 2 to 3 weeks after grafting demonstrated dysplastic differentiation of the HPV-16 and HPV-18 cell lines as characterized by abnormal mitotic figures, nuclear pleomorphism, and loss of basal polarity. RNA in situ hybridization confirmed the presenceof viral transcripts in the dysplastic grafts. G-banded chromosome analysis showed that both the HPV-16 and HPV-18 cell lines were aneuploid. Even after prolonged periods of implantation tumors did not form and invasion of underlying mouse stroma did not occur. Normal cervical cells differentiated normally and the C4-1 cell line formed tumors and demonstrated more pronounced nuclear abnormalities than the HPV immortalized grafts. Integration of HPV-16 and HPV-18 DNA into cervical cells leads to an indefinite growth capacity. These aneuploid cells differentiate abnormally in vivo, but do not form tumors or invade host tissue, consistent with a multistage theory of cervical carcinogenesis. o 1990 Academic press,

lomavirus. In vitro, human papillomavirus type 18 and 16 DNA immortalizes human cervical cells [3,4]. HPV gene expression is present both in immortalized cells and in established cervical carcinoma cell lines [3,.5]. Karyotype and DNA in situ analysis of cervical carcinoma cell lines has demonstrated human papillomavirus to be integrated in the host chromosomes at fragile sites and near oncogenes [6,7]. Integration of viral DNA may occur prior to alterations in chromosome constitution, suggesting that this process is an early step in cell immortalization and subsequent carcinogenesis [8]. This paper presents a description of an in vitro-in viva model for studying the influence of HPV 16 and 18 on in vivo differentiation of cervical epithelial cells. The study seeks to answer the following questions: Do cervical cells immortalized in vitro with HPV DNA implicated in cervical cancer demonstrate dysplastic differentiation in vivo? Is HPV gene expression associated with this process? Are the chromosome characteristics of cervical cells immortalized with human papillomavirus type 16 and 18 DNA similar to those of established cervical carcinoma cell lines?

I~C.

INTRODUCTION

MATERIALS

AND METHODS

Cell Culture

(HPVs) are implicated in the development of lower genital tract neoplasia including cervical carcinoma [1,2]. The precise nature of this association remains undetermined; however, one mechanism may be an alteration in normal cellular differentiation following infection with oncogenic human papilHuman papillomaviruses

Presented at the annual meeting of the Society of Gynecologic cologists, San Francisco, CA, February 4-7, 1990.

AND

On-

Normal human exocervical epithelial cells were derived from cervical specimens obtained at the time of hysterectomy for benign uterine conditions (leiomyomata, endometriosis, etc.), and shown to be free of dysplasia by histologic analysis. The cells were grown in MCDBl53-LB medium as described previously [9]. Cervical cells immortalized with HPV-16 (cell line CX16.2) or HPV 18 (cell line CX-18.1) DNA were obtained from established cell lines [3]. Cells of late passage (> 180

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0090-8258/90$1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ET AL.

in contact with the dorsal skin connective tissue of the mouse. Animals were sacrificed 2 to 3 weeks later and grafts were processed for histologic analysis. Assessment of Differentiation SECOND PLASTIC SHEET WfTbi INTACT CELL MOMO~AYER

FIRST PLASTIC

SHE IN-MUSCLE

fq.~f.’

Grafts were described as normal if they demonstrated epithelial stratification and basal polarity and were devoid of significant nuclear abnormalities. Grafts with one or more of the following features were classified as dysplastic: multinucleated cells, increased nuclear-to-cytoplasmic ratio, abnormal mitoses, or absence of basal cell polarity. Eight grafts from each cell line were analyzed including normal cervical epithelial cells cultured from three different women. In Situ Hybridization Graft sections were mounted on 3-aminopropyltriethoxysilane-coated slides and subjected to in situ hybridization with whole-genomic tritium-labeled antisense RNA probes as previously described [ 151. Positive controls included human tissues known to express human

FIG. 1. Diagram illustrating the technique epithelial monlayers into athymic mice.

for implanting

cervical

population doubhngs after HPV transfection) were used. Cell lines were maintained in serum-free, low-calcium MCDB 153-LB medium. C4-1 [lo], a cervical cancer cell line containing integrated HPV-18 DNA [ 1I], was maintained in DMEM/F12 medium supplemented with 5% fetal bovine serum. In vitro squamous differentiation was induced in cultures of HPV immortalized cell lines by allowing cells to become confluent and then increasing the calcium concentration of the medium from 0.1 to 2.0 mM [12]. Transplantation to Nude Mice When HPV DNA immortalized cells became confluent, the calcium concentration in the medium was increased to 2.0 mM for 3-5 days to induce intracellular bridging and adhesiveness [ 131.Intact monolayers of normal, HPV-16, HPV-18, and C4-1 cell lines were then removed from the tissue culture dish by digestion with dispase (2 mg/ml, Boehringer Mannheim) at 37°C for 60 min. Epithelial monolayers were transplanted into 6- to g-week-old female nude mice (m/m bg’/bg’) under a dorsal skin flap [14] (Fig. 1). Orientation of the graft was such that the basal side of the cervical epithelium was

FIG. grafted human taneous

2. Orientation of normal human exocervical epithelial cells under a dorsal skin flap into an athymic mouse. From the top: cervical epithelium, mouse connective tissue, muscle, subcufat with hair follicles, dermis, epidermis (X 75).

DIFFERENTIATION

OF HPV IMMORTALIZED

CERVICAL

CELLS

409

FIGS. 3,4. Graft of cervical cell line immortalized with HPV-18 (Fig. 3) or HPV-16 (Fig. 4) DNA and showing loss of basal polarity, abnormal stratification, nuclear pleomorphism, and increased nuclear-to-cytoplasmic ratio. Mitotic figures are seen well above the basal iaye of the graft ( x 150).

papillomavirus type 16 and 18 RNA. Negative controls included adjacent sections of grafts probed with antisense strands of the HPV genome as well as normal cervical cell grafts probed with HPV RNA. Chromosome Analysis HPV cell lines were subcultured 1:5 after reaching confluence and used for chromosome analysis 24 hr later. Colcemid (2 x 10’ M) was added and 4 hr later the medium was replaced with 0.075 M KCI. Cells were detached, then fixed in methanol/acetic acid (3/l). Gbanded chromosomes were obtained using the standard trypsin-Giemsa method. Chromosomes from each cell line were counted in 50 metaphases. Twenty karyotypes from sharp banding metaphases were analyzed for each cell line. RESULTS Grafts of human cervical epithelium were distinguishable from the overlying mouse skin by the absence of skin appendages such as hair follicles and dermal papillae. The mouse subdermal striated muscle was in closer proximity to the human epithelium than the mouse surface epithelium (Fig. 2). Grafted normal human ex-

ocervical cells had formed differentiated stratified epithelia 2 to 3 weeks following implantation. In contrast to grafted foreskin epithelium [ 141, human cervical cells did not produce an extensive upper layer of keratinized cells. Grafts of cell lines immortalized with HPV-16 and HPV-18 DNA disclosed findings resembling cervical intraepithelial neoplasia (CIN) (Figs. 3 and 4). The epithelial layers demonstrated both nuclear and architectural abnormalities, including loss of normal stratification and basal polarity, abnormal mitotic figures, nuclear pleomorphism, and an increased nuclear-to-cytoplasmic ratio. No invasion of the underlying mouse connective tissue was seen, and tumors did not form, even when cells were injected subcutaneously and observed for 1 year. The C4-1 cervical carcinoma cell line monolayers proliferated at a faster rate than the HPV immortalized lines and developed tumors as quickly as 3 weeks after implantation. Nuclear abnormalities appeared to be more pronounced than in the HPV immortalized grafts. In Situ Hybridization In situ hybridization was used to detect HPV gene expression in grafts of the HPV immortalized cell lines. Both the HPV-18 and HPV-16 immortalized cell line grafts expressed HPV RNA; however, HPV-18 grafts

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FIGS. 5,6. RNA in situ hybridization of HPV gene expression in the grafts derived from HPV-18 (Fig. 5) and HPV-16 (Fig. 6) cell lines. Positive signal (as demonstrated by white silver grains) is noted throughout the full thickness of the grafts.

tended to show a stronger in situ hybridization signal (Figs. 5 and 6). The normal cervical epithelial grafts did not show evidence of HPV gene expression.

DISCUSSION Normal cervical cells differentiate normally in this in vivo mouse model; however, cervical cells immortalized with human papillomavirus type 16 or 18 DNA, both of which have been associated with cervical cancer and its precursors, differentiate dysplastically. In this system, the HPV grafts demonstrate cytologic and architectural features of CIN, and underlying mouse connective tissue is not invaded and tumors do not form, even when grafts are left in place for 8 weeks. This is in contrast to the C4-1 cervical cancer cell line, which rapidly forms tumors and, when injected subcutaneously, vitro-in

Chromosome Analysis

G-banded karyotypes were obtained from the HPV-18 and HPV-16 cell lines (Figs. 7 and 8). Each cell line had an abnormal chromosome constitution consisting of numerical and structural alterations. This was a result of chromosome deletions, inversions, translocations, or partial duplications, features common to cervical carcinoma cell lines including C4-1 [ 16,171.

DIFFERENTIATION

OF HPV IMMORTALIZED

CERVICAL

:. 1 22

8

Ml

CELLS

411

ii! x

x

M4

FIGS. 73. Karyotypes of HPV-I8 (Fig. 7) and HPV-16 (Fig. 8) cell lines. Cell lines exhibit numerical and structural abnormalities due to translocations. deletions, or partial duplications. Consistent alterations are indicated by arrows or placed separately (M chromosomes).

will invade surrounding mouse tissue. Previously, other investigators have shown that cervical cell lines immortalized with HPV-16 DNA, when grown in an in vitro collagen raft culture system, demonstrate dysplastic morphology [4, I81 and invasion of the underlying basement membrane occurs early [4]. The in vivo model described in the present study probably more closely depicts what occurs in women with CIN, as natural progression to invasive cancer is usually a slow, infrequent process. Human papillomavirus gene expression is associated

with viral DNA integration into the host genome. This was confirmed in vitro during the development of the immortalized cell lines used for this experiment [3]. Not surprisingly, as shown by RNA in situ hybridization, evidence of HPV gene expression persists in this in viva model. In situ positivity is noted throughout the full thickness of the dysplastic grafts which may correlate with the lack of cellular differentiation as the cells stratified. In established cervical cancer cell lines [7,8], including

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C4- 1 [ 171,integration of viral DNA occurs at fragile sites, regions on chromosomes that are prone to breakage and often carry neighboring genes which are responsible for growth regulation or differentiation [ 191. Integration of oncogenic HPV DNA probably precedes development of an abnormal chromosome constitution [8]. The human papillomavirus type 16 and 18 cell lines used in this experiment are aneuploid and the integration site of HPV DNA in the HPV-16 cell line used in this study occurs at a site neighboring the ets-2 proto-oncogene on chromosome 21 [20]. However, in the HPV-16 and HPV-18 cell lines, aneuploidy itself, while conferring immortality, is not sufficient to render the cells malignant. This is consistent with the “multistep” theory of carcinogenesis [20,21] whereby integration of viral DNA and subsequent cell immortality allow for exposure to an additional insult or insults which would then be accompanied by a further genomic change and development of a frank malignant phenotype. These additional insults are probably not unique, and may include exposure to cigarette smoke byproducts [22], sex hormones [23], or an inherent genetic predisposition [24]. In conclusion, this study shows that cervical epithelial cells immortalized in vitro with oncogenic HPV DNA differentiate dysplastically in vivo. These aneuploid cells are not frankly malignant, and probably require an additional factor to render them cancerous. ACKNOWLEDGMENT The authors acknowledge and thank Dr. N. Popescu for karyotype analysis of the HPV-18 and -16 transfected cell lines.

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W., Lancaster, W. D., and DiPaolo, J. A. Characterization of normal human exocervical epithelial cells immortalized in vitro by papillomavirus types 16 and 18 DNA, Cancer Res. 48, 4620-4628 (1988). 4. Pecoraro, G., Morgan, D., and Defendi, V. Differential effects of human papillomavirus type 6, 16, and 18 DNA’s on immortalization and transformation of human cervical epithelial cells, Proc. Natl. Acad. Sci. USA 86, 563-567 (1989). 5. Schwarz, E., Freese, U. K., Gissman, L., Mayer, W., Roggenbock, B., Stremlau, A., and zur Hausen, H. Structure and transcription of human papillomavirus sequences in cervical carcinoma cells, Nature (London) 314, 111 (1985). 6. Durst, M., Croce, C. M., Gissmann, L., Schwarz, E., and Huebner, K. Papillomavirus sequences integrate near cellular oncogenes in some cervical carcinomas, Proc. Nat/. Acad. Sci. USA 84, 10701074 (1987).

7. Popescu, N. C., and DiPaolo, J. A. Preferential sites for viral integration on mammalian genome, Cancer Genet. Cytogenet. 42, 157-171 (1989). 8. Popescu, N. C., Amsbaugh, S. C., and DiPaolo, J. A. Human pap-

illomavirus type 18 DNA is integrated at a single chromosome site in cervical cancer cell line SW756, 1. Virol. 51, 1682-1685 (1987). 9. Pirisi, L.,Yasumoto, S., Feller, M., Doniger, J., and DiPaolo, J. A. Transformation of human fibroblasts and keratinocytes with human papillomavirus type 16 DNA, J. Virol. 61, 1061-1066 (1987). 10. Auersperg, N., and Hawryluk, A. F. Chromosome observations on three epithelial cell cultures derived from carcinomas of the human cervix, J. Natl. Cancer Inst. 28, 605-627 (1962). 11. Brandt, C. R., McDougall, J. K., and Galloway, D. A. Synergistic interactions between human papillomavirus type 18 sequences, herpes simplex virus infection, and chemical carcinogen treatment, in Cancer cells S/Papillomaviruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 179-186 (1987). 12. Hennings, H., Michael, D., Cheng, C., et al. Calcium regulation of growth and differentiation of mouse epidermal cells in culture, Cell 19, 245-254 (1980). 13. Pillai, S., Bilke, D. D., Hincenbergs, M., and Elias, P. M. Biochemical and morphological characterization and growth and differentiation of normal human neonatal keratinocytes in a serumfree medium, J. Cell Physiol. 134, 229-254 (1988). 14. Barrandon, Y., Li, V., and Green, H. New techniques for the grafting of cultured human epidermal cells onto athymic animals, J. Invest. Dermatol. 91, 315-318 (1988). 15. Staler, M. H., and Broker, T. R. In-situ hybridization detection of human papillomavirus DNA and messenger RNA in anogenital condylomas and a cervical carcinoma, Hum. Pathol. 17, 12501259 (1986). 16. Atkin, N. B. Chromosome changes in preneoplastic and neoplastic genital lesions, in Viral etiology of cervical cancer (R. Peto and H. zur Hausen, Eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 303-310 (1986). 17. James, G. K., Kalousek, D. K., and Auersperg, N. Karyotypic analysis of two related cervical carcinoma cell lines that contain human papillomavirus type 18 DNA and express divergent differentiation, Cancer Genet. Cytogenet. 38, 53-60 (1989). 18. McCance, D. J., Kopan, R., Fuchs, E., and Laimins, L. A. Human papillomavirus type 16 alters human epithelial cell differentiation in vitro, Proc. Natl. Acad. Sci. USA 8.5, 7169-7163 (1988). 19. Yunis, J. J., and Sureng, A. L. Constitutive fragile sites and cancer, Science 226, 1199-1204 (1987). 20. DiPaolo, J. A., Woodworth, C. D., Popescu, N. C., Notario, V., and Doniger, J. Induction of human squamous cell carcinoma by sequential transfection with human papillomavirus 16 DNA and viral Harvey ras, Oncogene 4, 395-399 (1989). 21. Munoz, N., Bosch, X., and Kaldor, J. M. Does human papillomavirus cause cervical cancer? The state of the epidemiological evidence, Brit. J. Cancer 57, l-5 (1988). 22. Slattery, M. L., Robison, C. M., Schuman, K. L., French, T. K., Abbott, T. M., Overall, J. C., Jr., and Gardner, J. W. Cigarette smoking and exposure to passive smoke are risk factors for cervical cancer, J. Amer. Med. Assoc. 261, 1593 (1989). 23. Beral, V., Hannaford, P., and Kay, C. Oral contraceptive use and malignancies of the genital tract: Results from the Royal College of General Practitioner’s Oral Contraception Study, Lancer 2, 1331 (1988). 24. Furgyik, S., Grubb, R., Kullander, S., Sandahl, B., Winegerup,

L., and Eydal, A. Familial occurrence of cervical cancer, stage O-IV, Acta Obstet. Gynecol. Stand. 65, 223-227 (1986).

Human cervical cells immortalized in vitro with oncogenic human papillomavirus DNA differentiate dysplastically in vivo.

Human papillomavirus (HPV) types 16 and 18 are associated with cervical dysplasia and carcinoma. In vitro integration of HPV-16 or HPV-18 DNA into cul...
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