American Journal of Pathology, Vol. 140, No. 2, February 1992 Copyright © American Association of Pathologists

Rapid Communication Human Papillomavirus 16 DNA Immortalizes Two Types of Normal Human Epithelial Cells of the Uterine Cervix Kouichiro Tsutsumi, Narasimhaswamy Belaguli, Sun Qi, Thomas 1. Michalak, Wayne P. Gulliver, Alan Pater, and Mary M. Pater From the Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada

Premalignant cervical lesions occur at the squamocolumnar junction and in endocervical epithelium and squamous ectocervical epithelium, in descending order of frequency. However, previously only ectocervical cells have been clearly shown to be immortalized in vitro by the oncogenic human papillomaviruses (HPVs). This report describes the immortalization of normal human ecto- and endocervical epithelial cells by the intact HPV 16 genome. Ectocervical epithelial cells (HEC) became immortalized (HEC-16) without crisis while endocervical cells (HEN) were immortalized (HEN-16) after undergoing crisis. HEN-16 and HEC-16 contained integrated HPV 16 DNA, expressed E6 and E7 mRNA, and were aneuploid and nontumorigenic. They also expressed cytokeratins in apattern similar to their distinct normal parental cells. These results suggest that both squamous and simple epithelial cells of uterine cervix are targets for immortalization by HPV 16 (Am JPathol 1992, 140:255-261)

The normal uterine cervix is composed of two distinct regions: the ectocervix, covered by squamous epithelium, and the endocervix, lined with simple columnar epithelium. As a result of squamous metaplasia, the endocervical epithelium is replaced by immature squamous epithelium which eventually becomes mature squamous epithelium.' This squamous metaplastic area, surrounded entirely by endocervical epithelium, is the com-

mon site for development of cervical cancer.2 In recent years, a large body of data has accumulated implicating certain human papillomavirus (HPV) types in the etiology of this cancer.3 HPV 16 has been detected in both squamous cell carcinomas and adenocarcinomas of the cervix.4 The same HPV is present and expressed in both cervical intraepithelial neoplasia (CIN) and adenocarcinoma in situ of the cervix.57 Woodworth et a18 and Pecoraro et a19 have recently reported the immortalization of human cervical epithelial cells by transfection with HPV 16 or HPV 18 DNA. Their immortalized cells exhibit characteristics of a squamous phenotype.810 However, since these cells were prepared from the region of the squamo-columnar junction between the ectocervix and endocervix,89 the exact target of HPV genome expression remains unclear. The aim of our experiments was to develop a model for studying the interaction between HPV 16 and simple epithelial cells from the endocervix. This report demonstrates that normal human endocervical epithelial cells can be immortalized in vitro with the full-length HPV 16 genome under the control of its own enhancer/promoter.

Materials and Methods Cervical Epithelial Cell Culture Human endocervical cell (HEN) and ectocervical cell (HEC) cultures were derived from a single cervical specimen obtained from hysterectomy (donor age, 44 years) which had been shown to be free of CIN by histologic examination. The squamous metaplastic cells of the Supported by the Medical Research Council of Canada and the National Cancer Institute of Canada. Accepted for publication November 26, 1991. Address reprint requests to Dr. Mary M. Pater, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada, Al B 3V6.

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transformation zone were identified microscopically and excised at a large distance from sampled ectocervical and endocervical epithelia. The large distance was reconfirmed by histologic identification of ample ecto- and endocervical tissues on both edges of the excised transformation zone tissue. The endocervical tissue, readily distinguishable by its color, and the ectocervical tissue were sectioned from the remaining specimen. The methods of Turyk et a'1 and Boyce and Ham,12 were used for culturing HEN and HEC, respectively. All cell cultures were maintained in keratinocyte growth medium (KGM, Clonetics, San Diego, CA).

Transfection HEC cells were electroporated according to Schlegel et al13 with some modifications. Cell suspensions (3 x 1 06 cells/ml) were mixed with 20 ,ug of BamHl linearized HPV 16 or 1 1 DNA and pulsed with 875 V/cm, 1 130 ,F. Lipofection was according to the method of Lu et al14

CK2 were purchased from Boehringer Mannheim, Mannheim, Germany and others were purchased from Sigma Chemical Co., St. Louis, Missouri. Vimentin mAb V9 was purchased from Sigma and fibronectin mAb 3E3 from Boehringer Mannheim. In the second layer, goat anti-mouse IgG reagent conjugated with fluorescein isothiocyanate was employed. The staining was evaluated with a Leitz Diaplan microscope.

Cytogenetic Analysis and Tumorigenicity Assays Chromosomes were prepared according to Pei et al19 Briefly, metaphase chromosome spreads were prepared, treated with 0.1-0.5 ,ug/ml colcemid, harvested, and pelleted. The cell pellet was resuspended in 0.075M KCI for 15 minutes fixed in three 30-minute changes of methanol/glacial acetic acid (3:1) at 220, spread on prewashed slides and air dried. For each cell type, two or three nude mice were injected with 3 x 1 06 cells at each of three sites and monitored for tumor formation for more than 4 months and 3 months for clonal cells.

Southern and Northern Blotting Results High molecular weight DNAs and total cell RNAs were prepared and analyzed as previously described.5'15'16 Blots were hybridized to nick-translated 32P-labelled HPV 16 DNA under stringent conditions and washed as previously described.515'16

RNase Protection Assay RNase protection assays were performed according to Kreig and Melton.17 Nt 501-863 fragment of HPV 16, which is homologous to a region of cell transcripts coding for E6 and E7, cloned into the vector Bluescript KS + (Stratagene, La Jolla, CA) served as a template for the synthesis of uniformly labelled sense and antisense probes for E6 and E7 mRNA. Five ,ug of total cellular RNA was hybridized with 100,000 cpm of the probe at 560C for 14 hours and digested with 700 U/ml of RNase Ti. RNase resistant hybrids were resolved in a 5% denaturing polyacrylamide sequencing gel and autoradiographed.

Immunofluorescence Staining Indirect immunofluorescence assays were performed according to Bartek et al18 Mouse monoclonal antibodies (mAbs) against cytokeratins (CKs), vimentin and fibronectin were used in the first layer. MAbs Ks13.1 and

Immortalization of Human Endo- and Ectocervical Epithelial Cells by HPV 16 DNA The pUC19 and HPV 11 DNA-transfected HEN and HEC became senescent at passages 5 and 7, respectively. HPV 16 immortalized cultures, HEN-16 and HEC-16, were selected by subculturing transfected cells that continued to proliferate. The frequency of immortalization by electroporation was four of nine experiments or 44% for HEC and 0 of 6 experiments for HEN. Therefore, HEN were transfected by lipofection and the frequency of immortalization was two of three experiments. HEC-1 6 grew continuously, whereas HEN-1 6 entered a 2-month period of crisis after passage 8. When this culture was passed at high cell density, a small number of dividing cells survived and these could be subcultured repeatedly. Both HEN-16 and HEC-16 have been in culture for more than 10 months with no signs of senescence. Since antibiotic resistance markers were not used for selection of HEN16 and HEC-16, these cells were killed within 7 days of incubation with 50 ,ug/ml G418, making them useful potential recipients for other genes.

Presence and Expression of HPV 16 DNA HEN-16 and HEC-16 contained HPV 16 DNA integrated into the cellular genome at different sites (Figure 1 A). The

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integrated viral DNAs were transcriptionally active and each gave two transcripts of similar 2.3 and 4.5 kb sizes, although transcripts were apparently more abundant in HEC-1 6 (Figure 1 B). Expression of mRNA for the HPV oncoproteins E6 and E7 was analyzed by RNase protection assays. Comparable levels of protection of the antisense probe was seen with RNA from both HEN-1 6 and HEC-16. No antisense RNA could be detected in either cell line (Figure 1C).

Figure 1. Presence and expression ofHPV 16 DNA in HEN-16 and HEC-16 A: Southern blotting. Cellular DNA (5 pg) from HEN-16 (lanes 1, 4, 6) and HEC-16(lanes 2, 5, 7) and HPV 16 DNA (lanes 3, 8) were digested with BamHI (lanes 1, 2, 3), Xbal (lanes 4, 5) or BamHIplus PstI (lanes 6, 7, 8) and hybridized with full-length HPV 16 DNA probe. B: Northem blotting. Total cellular RNA (10 pig) was from HEN-16 (lane 1), HEC-16 (lane 2) or HPV negative cervical cell line C33A (lane 3). Molecular weight markers are indicated on the right and, in descending order, are 4.5, 2.3 and 1.5 kilobases, respectively. C: RNase protection assaysfor HPV 16E6 and E7 RNA Lanes 1, 2 and 13 are for antisense probe, sense probe and molecular weight markers of 527, 429, 404 and 309 bases in descending order, respectively. Other odd and even numbered lanes are for assays with the antisense and sense probes, respectively. Lanes 3, 4: CaSki; lanes 5, 6: HEC-16; lanes 7, 8: HEN-16; lanes 9, 10: HT-3 HPV negative epithelial cell line; lanes 11, 12: tRNA

keratinocyte-like morphology (Figure 2A-a), whereas other cells had a pleomorphic epithelial appearance (Figure 2A-b). HEN-16 (Figure 2E) morphologically resembled HEN, composed of monolayers of keratinocyte-like cells with a few pleomorphic epithelial cells (Figure 2C). HEC (Figure 2B,D) and HEC-16 (Figure 1F) were composed of typical keratinocyte-like polygonal cells forming a cobblestone monolayer. Near-confluent HEC-1 6 were larger and flatter than HEC and were also clearly distinguishable from HEN-1 6.

In Vitro Morphology

Immunofluorescence Characterization In agreement with reports by others,1'120 the initial outgrowth in HEN cultures was morphologically distinguishable from that of HEC cultures (Figure 2). HEN with two distinct morphologies were observed. Some cells had a

The results for cytokeratin (CK) expression are shown in Figure 3A-D for HEN-16 and HEC-16 and are summarized in Table 1 for all cells. We observed diversity in the

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Figure 2. Morphology of HEN, HEC, HEN-16 and HEC-16 Bar = 100 ILm. A: PrImary culture of HEN. A-a: Keratinocyte-like cells. A-b: Pleomorphic epithelial cells. B: Primay culture of HEC. C: Seconda?y culture of near-confluent HEN. D: Secondary culture of nearconfluent HEC. E: Near-confluent HEN-1 6. These resemble secondary cultures ofHEN. F: Near-confluent HEC- 16. These are larger andflatter than the HEC and HEN-16

expression of CK using different mAbs specific for CK1 3 or CK18. HEN-16 and HEC-16 expressed CK13 and CK18 homogeneously, as detected by mAbs KS-1A3 and CY-90, respectively (Figure 3, C,D, Table 1). However, two mAbs specific for CK1 3 and one mAb specific for CK18 could distinguish between HEN-16 and HEC16, as well as HEN and HEC. Using mAb K8.12 for CK1 3 + 16 or Ks13.1 for CK13, HEC and HEC-16 showed promine'nt but heterogeneous staining (Figure 3A-b, Table 1) whereas HEN and HEN-1 6 were negative (Figure 3A-a, Table 1). In addition, using mAb CK2 specific for CK1 8, HEN and HEN-1 6 were positive (Figure 3B-a, Table 1) while HEC and HEC-1 6 were negative (Figure 3B-

b, Table 1). All cell strains expressed type 11 CKs (5, 6, 7, 8) and CK1 9 homogeneously. Staining of HEN with mAb KS-1 A3 against CK1 3 showed a heterogeneous pattern (Table 1). However, this antibody stained all HEN-16 cells, regardless of morphology (Figure 3C-a). HEN-16 and HEC-16, as well as their parental cells, showed a positive staining pattern with mAbs against vimentin and fibronectin (data not shown).

Cytogenetic Analysis and Tumorigenicity The parental HEN and HEC were predominantly diploid, as expected. The number of chromosomes in 1 00 each

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Figure 3. Identification of CKs in HEN-16(a) and HEC-16 (b) by immunofluorescence. Exposure time andprinting conditions were identical for all photographs. Bars = 50 p.m. A: K8. 12 mAb for CKsI3 and 16, shouwing nonstaining HEN-16 and heterogeneously positive HEC-16. B: CK2 mzAb for CK18, demonstrating homogeneouslyt positive HEN- 16 and nonstaining HEC-16. C,D: KS-1A3 mAbfor CK13 (C) and CY-90 mAb for CK18 (D).

Analyses of Clonal Cell Lines

of HEN-16 and HEC-16 were counted after passage in culture for 4 and 8 months. All cells were aneuploid at both times with approximately 50% hypotetraploid, 8% hypodiploid and 15% hypertetraploid. Nodules appeared after 3 weeks for HEC-1 6 but were absent by 6 weeks and no tumors formed for HEN-16 or HEC-16, whereas the positive control, SiHa carcinoma cell line containing HPV 16 DNA,15 gave tumors within 4 weeks.

Since HEN-1 6 and HEC-1 6 demonstrated heterogeneity, clonal cells were selected after 4 months in culture, by culturing from single cells seeded into miniwells. These cells were examined for immunofluorescence staining, cytogenetic and tumorigenic properties. All the clonal cells were identical to HEN-1 6 and HEC-1 6.

Table 1. Immunofluorescence Detection of Cytokeratins in HEN, HEN-16, HEC, and HEC-16

Discussion

Antibody

Cell culture

The transformation zone of the cervix, in which the simple Name H EM HEN-16 HEC* HEC-1 6 epithelial endocervical cells merge with the squamous epithelial ectocervical cells, is the region where the maK8.13 + t+ + + 5,6,7,8, + _ _ _ t jority of HPV-associated benign, premalignant, and maK8.60 1O,11 K8.12 + /_l 13,16 +/lignant lesions arise. It has, however, been postulated Ksi 3.1 13 + //+ /that squamous cell carcinomas can originate by proliferKS-1 A3 13 H4 + + + of endocervical cells in the endocervix, distant from ation + CY-90 18 CK2 + 18 + the transformation zone. In support of the malignant poK4.62 19 + + + + tential of endocervical cells, 10% of the CIN lesions have * Cultures examined at p assage 2. been shown to be surrounded completely by endocervit + = homogeneously rpositive. tissue whereas only 3% were found in the ectocercal t - = homogeneously rnegative. cisli = 50 to 100% of cells positive. 11+/vix.21 However, in vitro studies with human cervical cells Cytokeratin specificity

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predating this report have not addressed whether both endo- and ectocervical cells are targets of HPV 16 oncogenicity. In this report we demonstrate for the first time that HPV 16 can immortalize human endocervical simple epithelial cells as well as ectocervical squamous epithelial cells. In agreement with reports by others for HPV 16 immortalized human keratinocytes,'91319 2224 the HPV 16 sequences were integrated into the cellular genome (Figure 2A), cells expressed viral RNAs (Figure 2B) and were aneuploid. The expected presence of E6 and E7 mRNA in HEN-16 and HEC-16 was also confirmed (Figure 2C). HEC-16 became immortalized without crisis, while crisis was observed for HEN-1 6. Since parental cells were prepared from tissues from the same individual, the occurrence of crisis probably was due to cell type-specific differences rather than genetic differences. The molecular basis of host cell specificity in response to HPV 16 remains to be elucidated. Band et a125 have described that HPV 16 immortalizes normal human mammary epithelial cells, although HPV 16 has not been reported to be associated with breast cancer. Since HEN might potentially contain any of four endocervical cell types,26 the exact origin of HEN-i6 remains unclear. However, based on our in vitro results (Table 1) and the in vivo studies by others, it appears likely that these cells were derived from undifferentiated reserve cell (RCs) in HEN. Levy et a127 have reported the specific staining of RCs with mAb KS-1A3 for CK1 3. Also in our in vitro studies, parental, probably heterogeneous HEN were heterogeneously stained and growth-selected HEN-1 6 were homogeneously stained by this mAb. For a second mAb (K8.12 for CK 13 + 16), both Gigi-Leitner et a128 and Ivany et al29 showed no staining of RCs; similarly, both HEN and HEN-16 were specifically negative (Table 1). Based on these observations as well as morphology, the endocervical cells can be clearly distinguished from the ectocervical cells and we suggest that HEN-16 is derived from RCs in HEN. The possibility that the tissue was taken from areas of focal squamous metaplasia cannot be conclusively excluded although the cervical tissue from which the cells were derived was pathologically normal and the primary HEN did not contain HPV 16 DNA (data not shown) and senesced after a few passages. Neoplastic transformation of cultured cells is believed to result from multiple cellular changes.30 Available evidence suggests that HPV 16 alone is not sufficient for the human epithelial cells to become tumorigenic8 1013,19,25 and HEN-1 6 and HEC-1 6 were negative for tumorigenicity. Therefore, the established, G418 sensitive HEN-16 and HEC-16 should permit detailed in vitro and in vivo studies on the role of oncogenes31'32 and steroid hor-

mones16'32' in the progression of these cells into the fully malignant phenotype.

Acknowledgments The authors thank M. Rahimtula for excellent technical assistance, H. zur Hausen and L. Gissmann for HPV 16 and 1 1 plasmids, and S. Atkins for typing the manuscript.

References 1. Ferenczy A, Winkler B: Anatomy and histology of the cervix. Blaustein's Pathology of the Female Genital Tract, 3rd ed. Edited by Kurman RJ. New York, Springer-Verlag, 1987, pp 141-157 2. Burghardt E: Natural history of cervical lesions. Banbury Report 21: Viral Etiology of Cervical Cancer. Edited by Peto R, and zur Hausen H. New York, Cold Spring Harbor Laboratory, 1986, 81-89 3. Howley PM, Schlegel R: The human papillomaviruses. An overview. Am J Med 1988, 85 (Suppl 2A):155-158 4. Tase T, Okagaki T, Clark BA, Manias DA, Ostrow RS, Twiggs LB, Faras AJ: Human papillomavirus types and localization in adenocarcinoma and adenosquamous carcinoma of the uterine cervix: A study by in situ DNA hybridization. Cancer Res 1988, 48:993-998 5. Pater MM, Dunne J, Hogan G, Ghatage P, Pater A: Human papillomavirus type 16 and 18 sequences in early cervical neoplasia. Virology 1986,155:13-18 6. Farnsworth A, Laverty C, Stoler MH: Human papillomavirus messenger RNA expression in adenocarcinoma in situ of the uterine cervix. Int J Gynecol Pathol 1989, 8:321-330 7. Crum CP, Symbula M, Ward BE: Topography of early HPV 16 transcription in high-grade genital precancers. Am J Pathol 1989, 134:1183-1188 8. Woodworth CD, Bowden PE, Doniger J, Pirisi L, Barnes W, Lancaster WD, DiPaolo JA: Characterization of normal human exocervical epithelial cells immortalized in vitro by papillomavirus types 16 and 18 DNA. Cancer Res 1988, 48:4600-4628 9. Pecoraro G, Morgan D, Defendi V: Differential effects of human papillomavirus type 6, 16 and 18 DNAs on immortalization and transformation of human cervical epithelial cells. Proc NatI Acad Sci USA 1989, 86:563-567 10. Woodworth CD, Waggoner S, Barnes W, Stoler MH, DiPaolo HA: Human cervical and foreskin epithelial cells immortalized by human papillomavirus DNAs exhibit dysplastic differentiation in vivo. Cancer Res 1990, 50:3709-3715 11. Turyk ME, Golub TR, Wood NB, Hawkins JL, Wilbanks GD: Growth and characterization of epithelial cells from normal human uterine ectocervix and endocervix. In Vitro 1989,

25:544-556 12. Boyce ST, Ham RG: Cultivation, frozen storage, and clonal growth of normal human epidermal keratinocytes in serumfree media. J Tiss Cult Meth 1985, 9:83-93.

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13. Schlegel R, Phelps WC, Zhang Y-L, Barbosa MS: Quantitative keratinocytes assay detects two biological activities of human papillomavirus DNA and identifies viral types associated with cervical carcinoma. EMBO J 1988, 7:3181-3187 14. Lu L, Zeitlin PL, Guggino WB, Craig RW: Gene transfer by lipofection in rabbit and human secretory epithelial cells. Pflugers Arch 1989, 415:198-203 15. Pater MM, Pater A: Human papillomavirus types 16 and 18 sequences in carcinoma cell lines of the cervix. Virology 1985,145:313-318 16. Pater MM, Hughes GA, Hyslop DE, Nakshatri H, Pater A: Glucocorticoid-dependent oncogenic transformation by type 16 but not type 1 1 human papillomavirus DNA. Nature 1988, 335:832-835 17. Kreig PA, Melton DA: In vitro RNA synthesis with SP6 RNA polymerase. Meth Enzymol 1987,155:397-415 18. Bartek J, Bartkova J, Lalani E-N, Brezina V, TaylorPapadimitriou J: Selective immortalization of a phenotypically distinct epithelial cell type by microinjection of SV40 DNA into cultured human milk cells. Int J Cancer 1990, 19.

20.

21.

22.

23.

45:1105-1112 Pei XF, Gorman PA, Watt FM: Two strains of hluman keratinocytes transfected with HPV 16 DNA: compqrison with the normal parental cells. Carcinogenesis 1991, 12:277-284 Dixon IS, Stanley MA: Immunofluorescence studies of human cervical epithelial in vivo and in vitro using antibodies against specific keratin components. Mol Biol Med 1984, 2:37-51 Abdul-Karim FW, Fu YS, Reagon JW, Wentz WB: Morphometric study of intraepithelial neoplasia of the uterine cervix. Obstet Gynecol 1982, 60:210-217 Pirisi L, Yasumoto S, Feller M, Doniger J, DiPaolo JA: Transformation of human fibroblasts and keratinocytes with human papillomavirus type 16 DNA. J Virol 1987, 61:10611066 Durst M, Dzarlieva-Petrusevska RT, Boukamp P, Fusenig NE, Gissmann L: Molecular and cytogenetic analysis of immortalized human primary keratinocytes obtained after transfection with human papillomavirus type 16 DNA. Oncogene 1987,1:251-256

24. Hawley-Nelson P, Vousden KH, Hubbert NL, Lowy DR, Schiller JT: HPV 16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J 1989, 8:39053910 25. Band V, Zajchowski D, Kulesa V, Sager R: Human papillomavirus DNAs immortalize normal human mammary epithelial cells and reduce their growth requirements. Proc Natl Acad Sci USA 1990, 87:463-467 26. Kudo R, Sagae S, Hayakawa 0, Ito E, Horimoto E, Hashimoto M: Morphology of adenocarcinoma in situ and microinvasive adenocarcinoma of the uterine cervix. Acta Cytol 1991, 35:109-116 27. Levy R, Czernobilsky B, Geiger B: Subtyping of epithelial cells of normal and metaplastic human uterine cervix, using

28. 29.

30.

31.

32.

33.

polypeptide-specific cytokeratin antibodies. Differentiation 1988, 39:185-196 Gigi-Leitner 0, Geiger B, Levy R, Czernobilsky B: Cytokeratin expression in squamous metaplasia of the human uterine cervix. Differentiation 1986, 31:191-205 Ivanyi D, Groeneveld E, Doornewaard GV, Mooi AJ, Hageman PC: Keratin subtypes in carcinomas of the uterine cervix: Implications for histogenesis and differential diagnosis. Cancer Res 1990, 50:5143-5152 Barrett JC, Fletcher WF: Cellular and molecular mechanism of multistep carcinogenesis in cell culture models. Mechanism of Environmental Carcinogenesis: Multistep Models of Carcinogenesis Edited by Barrett JC. Boca Raton, FL, CRC Press, 1987, pp 73-116 DiPaolo JA, Woodworth CD, Popescu NC, Notario V, Doniger J: Induction of human cervical squamous cell carcinoma by sequential transfection with human papillomavirus 16 DNA and viral Harvey ras. Oncogene 1989, 4:395-399 Durst M, Gallahan D, Jay G, Rhim JS: Glucocorticoidenhanced neoplastic transformation of human keratinocytes by human papillomavirus type 16 and activated ras oncogene. Virology 1989,173:767-771 Pater A, Bayatpour M, Pater MM: Oncogenic transformation by human papillomavirus type 16 deoxyribonucleic acid in the presence of progesterone or progestins from oral contraceptives. Am J Obstet Gynecol 1990,162:1099-i 103

Human papillomavirus 16 DNA immortalizes two types of normal human epithelial cells of the uterine cervix.

Premalignant cervical lesions occur at the squamo-columnar junction and in endocervical epithelium and squamous ectocervical epithelium, in descending...
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