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Rapid dysplastic transformation of human genital cells by human papillomavirus type 18
GYNECOLOGIC
ONCOLOGY
38, 343-346 (1990)
Rapid Dysplastic Transformation of Human Genital Cells by Human Papillomavirus Type 18’ WILLARD BARNES M.D.,*,* CRAIG WOODWORTH, PH.D.,? STEVEN WAGGONER, M.D.,* MARK STOLER, A. BENNETT JENSON, M.D.,8 GREGORIO DELGADO, M.D.,* AND JOSEPH DIPAOLO, PH.D.t
M.D.,*
*Department of Obstetrics and Gynecology, $Department of Pathology, Georgetown University Medical Center, Washington, DC 20007; *Laboratory of Biology, Division of Cancer Etiology, National Cancer Institute, Bethesda, Muryland 20892; and fDepartment of Pathology, Cleveland Clinic Foundation, Clevelund, Ohio 44195 Received January 22, 1990
This study delineates differences in biologic activity between human papillomavirus (HPV) types 16 and 18. Human cervical and foreskin epithelial cells were cultured and transfected with recombinant HPV-16 and -18 DNA, resulting in immortalized cell lines. Normal epithelial cells as well as HPV-16 and -18 immortalized cells of both early passages(less than 40 population doublings) and late passages(greater than 180 population doublings) were transplanted in athymic mice. Normal squamous cells formed well-stratified epithelium, while HPV immortalized cells developed either normal-appearing epithelium or typical dysplastic changes. Dysplastic changes were seen in none of the 13 grafts with early-passage HPV-16 cell lines, while 9 of 14 grafts with early-passage HPV-18 cell lines developed dysplasias (P < 0.0004). These results support previous clinical observations suggesting that HPV-18 may be associated with a more aggressive and rapidly progressive form of cervical neoplasia. o IWO Academic Press, Inc.
INTRODUCTION The involvement
of an infectious,
sexually transmitted
factor in the development of cervical intraepithelial neoplasias and cancers has been suggested by epidemiologic studies [ 1,2]. Recently, much attention has been focused on the human papillomavirus (HPV) and its possible role in the development of benign and cancerous lesions of the genital tract. The cottontail rabbit papillomavirus has been shown to be oncogenic in its natural host [3], and human papillomaviruses have been demonstrated by electron microscopy and molecular hybridization to be ’ Supported in part by ACS Grant 89-13~0 and US PHS Grant CA43629. Presented at the annual meeting of the Society of Gynecologic Oncologists, San Francisco, CA, February 4-7, 1990. ’ To whom requests for reprints should be addressed.
present in genital intraepithelial neoplasias and cancers [4,5]. In addition, HPV has been demonstrated by molecular hybridization to be present in the primary cancer site as well as lymph node metastases [6]. HPV types 6, 11, 16, 18, 31, 33, and 35 have been found in cervical neoplasias [7-l 11.HPV-16 and -18 are the most common viral types found in cervical cancers, and HPV-6 and -11 are the most common viral types found in condyloma
[121. Because HPV-16 and -18 are the most common viral types associated with cervical cancers, several clinical, histopathologic studies have focused on determining possible differences in behavior of the associated genital neoplasias. In a previous study of 30 invasive cervical carcinomas, Barnes et al. reported an association between HPV-18 and grade 3 tumors, and in addition, the mean age of cancer patients with HPV-18 was 37 compared to 49 years for patients with HPV-16 [13]. More recently, Walker et al. in an analysis of 100 cervical cancers demonstrated that patients with HPV-18-containing tumors tended to have recurrences more frequently and were more likely to have a history of recent normal Papanicolaou smears [14]. The possible association of HPV-18 with a more rapidly progressive or aggressive group of genital neoplasias was also suggested by studies by Lorincz et al., who demonstrated that HPV-18 sequences were detected more commonly in invasive carcinomas (18%) than in intraepithelial neoplasias (1%) [15]. Kurman et al. also demonstrated a preponderance of HPV-18 in invasive carcinoma (22%) compared to intraepithelial neoplasias (3%), while HPV16 was seen in approximately equal frequencies in invasive carcinomas (41%) and intraepithelial neoplasias (37%) [16].
343 0090-8258190 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
344
BARNES ET AL.
TABLE 1 Histology of Grafts Derived from Normal and Transfected Epithelium Stratified Normal HPV-16 Early passage Late passage HPV-18 Early passage Late passage
Dysplastic
Total
29
0
29
13 4
0 19
13 23
5 3
9 9
14 12
section with dispace and placed on a rectangular sheet of Silastic [20]. The graft was placed subcutaneously on the dorsal musculature of female athymic mice. The animals were sacrificed after 2 to 3 weeks and the grafts were removed, stained with hematoxylin and eosin (H&E), and examined microscopically. Grafts with one or more of the following histologic features were clas-
Human papillomavirus has not to date been successfully cultured in the laboratory, making basic, controlled study of its interaction with human cells difficult. Woodworth et al. described an in vitro system for studying the interaction between HPV-16 and -18 recombinant DNA and normal cervical epithelial cells [17]. Transfection of epithelial cells with recombinant HPV-16 or -18 DNA resulted in immortalized cell lines containing either integrated HPV-16 or -18 sequences. Transplantation of monolayers of these immortalized cell lines to athymic mice has resulted in the development of histologically dysplastic epithelium [17]. The present study was undertaken to describe any differences between HPV-16 and -18 xenografts in the development of epithelial neoplasia utilizing this model. MATERIALS AND METHODS Human cervical and foreskin epithelial cells were isolated, cultured, and grafted as previously described [17]. Tissue fragments were placed in MCDB153-LB culture medium containing 0.25% collagenase, and epithelial outgrowth occurred after 7 to 10 days. Secondary cultures at 40 to 60% confluence were transfected with the recombinant plasmid pMHPV16d or pSHPVlM containing the neomycin resistance gene and entire HPV genomes [17,18]. Immortalized cells were selected by resistance to G418 or by their ability to proliferate with continuous subculturing after normal epithelium had senesced. HPV immortalized cervical and foreskin epithelial cell lines of both early and late passage were used. Early passage refers to cells used prior to 40 population doublings after HPV transfection, and late passage refers to cells cultured for more than 180 population doublings. Nontransfected epithelial cells in secondary or tertiary cultures were utilized for control grafts. Squamous differentiation was induced in culture by allowing cells to become conFIG. 1. Morphologyof xenografts containing normal (A) and HPV fluent and increasing the calcium concentration to 2.0 immortalized cells (B,C). (A) Graft of normal human cervical cells mM to induce intracellular attachment and stratification showing stratified squamous epithelium; (B) graft of HPV-16 immor[17,19]. After further incubation for 3 to 5 days intact talized cervical cells transplanted at later passage; (C) graft of HPVmonolayers were removed from the culture dish by dis- 18 immortalized foreskin cells transplanted at early passage.
KA~IU v r 3rLA3’ “““Z I I\ TRANSFORMATION n ’ nrn -\lnnT
345
traepithelial stages has been suggested by Berkeley [24,25]. While the exact etiologic role of human papillomavirus in the genesis of cervical cancer is still unknown, a strong association of HPV-16 and HPV-18 to cervical cancer has been established. Questions have been raised regarding prognostic significance of HPV types and the possible association of HPV-18 with more biologically aggressive cancers [ 12,161. Other previous clinical studies demonstrate a difference in the observed frequency between HPV-16 and -18 in premalignant and malignant cervical lesions [IS, 161; the observation that RESULTS HPV-18 is much less likely to be seen in a dysplastic A total of 91 epithelial grafts were analyzed. The types lesion as compared to a cancerous lesion is consistent of cells grafted were as follows: normal epithelial cells, with the hypothesis that HPV-18 may be associated with 29; HPV-16 immortalized cells, 36; HPV-18 immortalized lesions that bypass or quickly progress through the incells, 26. Of the grafts utilizing HPV-16 immortalized traepithelial stages. In this study, both HPV-16 and -18 cells, 13 grafts were with early-passage cells and 23 grafts immortalized cell lines produced dysplastic epithelium with late-passage cells. In the HPV-18 immortalized when transplanted in athymic mice. Grafts composed of grafts, 14 utilized early-passage cells and 12 late-passage cultured, immortalized cells of greater than 180 popucells. Microscopic examination of H&E-stained grafts is lation doublings (late passage) formed dysplastic epithedetailed in Table 1 and Fig. 1. Normal epithelial cells lium in approximately equal numbers in HPV-16 and formed stratified squamous tissue in 29 grafts. Of the 36 -18 grafts; however, HPV-18 immortalized cells exhibited grafts utilizing the HPV-16 immortalized cells, 17 formed approximately the same ability to form dysplastic grafts normal-appearing stratified squamous epithelium and 19 utilizing cultured cells of less than 40 population doushowed dysplastic changes. None of the HPV-16 early- blings (early passage) as with late-passage grafts. In conpassage cells resulted in dysplastic changes, while 19 of trast, HPV-16 cell lines produced no dysplastic epithe24 late-passage cell grafts resulted in dysplastic changes. lium in grafts utilizing early-passage cells. This rapid Eighteen of the twenty-six HPV-18 immortalized cell dysplastic transformation of human genital cells by HPVgrafts showed dysplastic changes, with 9 of 14 early- 18 compared to HPV-16 in this in vitro/in vivo system passage grafts and 9 of 11 late-passage grafts showing is consistent with the previous clinical studies suggesting dysplasia. The difference in dysplastic grafts resulting an association between HPV-18 and rapidly progressive from early-passage cells in HPV-16 and -18 grafts is sta- cervical neoplasia. tistically significant (P < 0.0004). No invasion of the underlying basal membrane was observed in either HPVREFERENCES 16 or HPV-18 cell lines. Southern blotting was used to confirm the presence of 1. Rotkin, I. D. A comparison review of key epidemiological studies HPV 16 and 18 in transfected cell lines prior to grafting. in cervical cancer related to current searches for transmissible In addition, in situ hybridization was utilized to confirm agents, Cancer Res. 33 (1973). the presence of HPV messenger RNA in the grafts. Three 2. Kessler, 1. I. Venereal factors in human cervical cancer, Cancer HPV-18 immortalized cell lines which formed dysplastic 39, 1912-1919 (1977). epithelium at early passage expressed moderate to low 3. Lancaster, W. D., and Olson, C. Animal papillomaviruses, Milevels of HPV RNA. Two HPV-16 immortalized cell lines crobio/. Rev. 46, 191-207 (1982). which formed dysplastic epithelium at late passage ex4. Hills, E., and Laverty, C. R. Electron microscopic detection of pressed virus RNA at low to barely detectable levels. papillomavirus particles in selected koilocytotic cells in routine cervical smear, Acta Cytol. 23, 53-58 (1979). Grafts composed of C-41 cells had stronger in situ signals 5. Lancaster, W. D., Kurman, R. J., Sanz, L. E., and Jenson, A. B. than HPV- 16- and HPV- 18-derived grafts. sified as dysplastic: abnormal mitoses, multinucleated cells, increased nuclear-to-cytoplasmic ratio, and absence of basal cell polarity. Graft sections were also mounted on 3-aminopropyltriethoxysilane-coated slides and subjected to in situ to hybridization with wholegenomic tritium-labeled antisense RNA probes for HPV types 16 and 18 [21,22]. In addition, similar grafts using C-41 cervical carcinoma cells were subjected to in situ hybridization [23].
DISCUSSION The clinical presentation and disease course of patients with cervical cancer can vary greatly. As noted by Bain and Cracker, some individuals appear to have a rapid onset of cervical cancer, and a possible distinct subset of cancers with a rapid onset which bypasses the in-
Human papillomavirus: Detection of viral DNA sequences and evidence for molecular heterogeneity in metaplasias and dysplasias of the uterine cervix, Interviro/opy 20, 202-212 (1983). 6. Lancaster, W. D., Castellano, C., Santos, C., Delgado, G., Kurman, R. J., and Jenson, A. B. Human papillomavirus DNA in cervical carcinoma from primary and metastatic sites, Amer. J. Obster. Gynecol. 154, 115-l I8 (1986). 7. Durst, M., Gissmann, L., Ikenberg, H., and zur Hausen, H. A papillomavirus DNA from a cervical carcinoma and its prevalence
346
BARNES
in cancer biopsy samples from different Not/.
Acud.
8. Boshart, M., len, W.. and its presence from cervical
Sci.
USA
80, 3812-3815
geographic
regions, Prac.
(1983).
ET AL. role for type I8 in rapid progression, 159, 293-296 (1988).
Amer.
J. Obstet.
Gynecol.
Gissmann. L., Ikenberg, H., Kleinheinz. A.. Scheurzur Hausen, H. A new type of papillomavirus DNA. in genital cancer biopsies and in cell lines derived cancer, EMEO J. 3, 1151-l 157 (1984).
17. Woodworth, C.. Waggoner, S., Barnes, W., Stoler, M., DePaolo. J. Human cervical and foreskin epithelial cells immortalized by human papillomavirus DNA’s exhibit dysplastic differentiation in Vivo. Cancer Res. 50, 3709-3715 (1990).
9. Lorincz, A. T.. Lancaster, W. D.. and Temple, G. F. Cloning and characterization of the DNA of a new human papillomavirus from a woman with dysplasia of the uterine cervix, J. Viral. 58, 22% 229 (1986).
18. Woodworth, C. D., Bowden, P. E., Doniger. J.. Pirisi, L.. Barnes, 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, 4260-4268 (1988).
IO. de Villiers, E. M.. Gissmann, I... and zur Hausen, H. Molecular cloning of viral DNA from human genital warts, J. Viral. 40, 932935 (1981). Il.
Gissmann. L., Wolnik, L., Ikenberg, H.. Koldovsky. U.. Schnurch. H. G.. and zur Hausen, H. Human papillomavirus types 6 and I I DNA sequences in genital and laryngeal papillomas and in some cervical cancers, Proc. Natl. Acud. Sci. USA 80, 56O563 (1983).
19. Pillai, S., Bilke. D. D., Hincenbergs, M., and Elias, P. M. Biochemical and morphological characterization of growth and differentiation of normal human neonatal keratinocytes in a serum134, 229-237 (1988). free medium, J. Cell. Physiol. 20. Barrandon. Y., Li, D., and Green, H. J. New techniques for the grafting of cultured human epidermal cells onto athymic animals. Inve.st. Dermutol. 91, 315-318 (1988).
12. Reid, R.. Greenberg, M.. Jenson. A. B., et ul. Sexually transmitted papillomaviral infections. I. The anatomic distribution and pathologic grade of neoplastic lesions associated with different viral 156, 212 (1987). types. Amer. J. Oh.stet. Gynrcol.
21. 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. Puthol. 17, 12501259 (1986).
13. Barnes, W., Delgado, G., Kurman, R. J.. et (I/. Possible prognostic significance of human papillomavirus type in cervical cancer, Gynew/. Oncol. 29, 267 (1988).
22. Angerer.
L. M.. Staler. M. H., and Angerer. R. C. in In situ hyApplicutions to neurobiology (K. Valentino, J. Eberwine, and J. Barchus, Eds.). Oxford Univ. Press, New York. pp. 42-70 (1987). hridizution:
14. Walker, J., Bless. J. D.. Liao, S.. Berman. M.. Bergen, S.. and Wilczynski, S. Human papillomavirus genotype as a prognostic indicator in carcinoma of the uterine cervix. Ohstet. (;vneco/. 74, 781-785 (1989).
23. Auersperg. N.. and Hawryluk, A. F. Chromosome observations on three epithelial cultures derived from carcinomas of the cervix.
15. Lorincz, A., Temple. G. F.. Kurman. R. J.. Jenson, A. B.. and Lancaster, W. D. The oncogenic association of specific HPV types with cervical neoplasia, J. Nut/. Cancer Inst. 79, 671-677 (1987).
24. Bain. R. W., and Cracker, A. W. Rapid onset of cervical cancer in an upper socioeconomic group, Amer. J. Ohstet. G.vneco/. 146, 366-371 (1983).
16. Kurman. R. J., Schiffman. M. H.. Lancaster, W., Reid, R.. Jenson. A. B., Temple, G. F.. and Lorincz. A. T. Analysis of individual human papillomavirus types in cervical neoplasia: A possible
25. Berkeley, A. S.. LiVolsi, V. A., and Schwartz, P. E. Advanced squamous cell carcinoma of the cervix with recent normal Papanicolaou tests, Lancet 2, 375-376 (1980).
J. Nut/.
Cancer
Inst.
28, 605-627
(1987).
ONCOLOGY
38, 343-346 (1990)
Rapid Dysplastic Transformation of Human Genital Cells by Human Papillomavirus Type 18’ WILLARD BARNES M.D.,*,* CRAIG WOODWORTH, PH.D.,? STEVEN WAGGONER, M.D.,* MARK STOLER, A. BENNETT JENSON, M.D.,8 GREGORIO DELGADO, M.D.,* AND JOSEPH DIPAOLO, PH.D.t
M.D.,*
*Department of Obstetrics and Gynecology, $Department of Pathology, Georgetown University Medical Center, Washington, DC 20007; *Laboratory of Biology, Division of Cancer Etiology, National Cancer Institute, Bethesda, Muryland 20892; and fDepartment of Pathology, Cleveland Clinic Foundation, Clevelund, Ohio 44195 Received January 22, 1990
This study delineates differences in biologic activity between human papillomavirus (HPV) types 16 and 18. Human cervical and foreskin epithelial cells were cultured and transfected with recombinant HPV-16 and -18 DNA, resulting in immortalized cell lines. Normal epithelial cells as well as HPV-16 and -18 immortalized cells of both early passages(less than 40 population doublings) and late passages(greater than 180 population doublings) were transplanted in athymic mice. Normal squamous cells formed well-stratified epithelium, while HPV immortalized cells developed either normal-appearing epithelium or typical dysplastic changes. Dysplastic changes were seen in none of the 13 grafts with early-passage HPV-16 cell lines, while 9 of 14 grafts with early-passage HPV-18 cell lines developed dysplasias (P < 0.0004). These results support previous clinical observations suggesting that HPV-18 may be associated with a more aggressive and rapidly progressive form of cervical neoplasia. o IWO Academic Press, Inc.
INTRODUCTION The involvement
of an infectious,
sexually transmitted
factor in the development of cervical intraepithelial neoplasias and cancers has been suggested by epidemiologic studies [ 1,2]. Recently, much attention has been focused on the human papillomavirus (HPV) and its possible role in the development of benign and cancerous lesions of the genital tract. The cottontail rabbit papillomavirus has been shown to be oncogenic in its natural host [3], and human papillomaviruses have been demonstrated by electron microscopy and molecular hybridization to be ’ Supported in part by ACS Grant 89-13~0 and US PHS Grant CA43629. Presented at the annual meeting of the Society of Gynecologic Oncologists, San Francisco, CA, February 4-7, 1990. ’ To whom requests for reprints should be addressed.
present in genital intraepithelial neoplasias and cancers [4,5]. In addition, HPV has been demonstrated by molecular hybridization to be present in the primary cancer site as well as lymph node metastases [6]. HPV types 6, 11, 16, 18, 31, 33, and 35 have been found in cervical neoplasias [7-l 11.HPV-16 and -18 are the most common viral types found in cervical cancers, and HPV-6 and -11 are the most common viral types found in condyloma
[121. Because HPV-16 and -18 are the most common viral types associated with cervical cancers, several clinical, histopathologic studies have focused on determining possible differences in behavior of the associated genital neoplasias. In a previous study of 30 invasive cervical carcinomas, Barnes et al. reported an association between HPV-18 and grade 3 tumors, and in addition, the mean age of cancer patients with HPV-18 was 37 compared to 49 years for patients with HPV-16 [13]. More recently, Walker et al. in an analysis of 100 cervical cancers demonstrated that patients with HPV-18-containing tumors tended to have recurrences more frequently and were more likely to have a history of recent normal Papanicolaou smears [14]. The possible association of HPV-18 with a more rapidly progressive or aggressive group of genital neoplasias was also suggested by studies by Lorincz et al., who demonstrated that HPV-18 sequences were detected more commonly in invasive carcinomas (18%) than in intraepithelial neoplasias (1%) [15]. Kurman et al. also demonstrated a preponderance of HPV-18 in invasive carcinoma (22%) compared to intraepithelial neoplasias (3%), while HPV16 was seen in approximately equal frequencies in invasive carcinomas (41%) and intraepithelial neoplasias (37%) [16].
343 0090-8258190 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
344
BARNES ET AL.
TABLE 1 Histology of Grafts Derived from Normal and Transfected Epithelium Stratified Normal HPV-16 Early passage Late passage HPV-18 Early passage Late passage
Dysplastic
Total
29
0
29
13 4
0 19
13 23
5 3
9 9
14 12
section with dispace and placed on a rectangular sheet of Silastic [20]. The graft was placed subcutaneously on the dorsal musculature of female athymic mice. The animals were sacrificed after 2 to 3 weeks and the grafts were removed, stained with hematoxylin and eosin (H&E), and examined microscopically. Grafts with one or more of the following histologic features were clas-
Human papillomavirus has not to date been successfully cultured in the laboratory, making basic, controlled study of its interaction with human cells difficult. Woodworth et al. described an in vitro system for studying the interaction between HPV-16 and -18 recombinant DNA and normal cervical epithelial cells [17]. Transfection of epithelial cells with recombinant HPV-16 or -18 DNA resulted in immortalized cell lines containing either integrated HPV-16 or -18 sequences. Transplantation of monolayers of these immortalized cell lines to athymic mice has resulted in the development of histologically dysplastic epithelium [17]. The present study was undertaken to describe any differences between HPV-16 and -18 xenografts in the development of epithelial neoplasia utilizing this model. MATERIALS AND METHODS Human cervical and foreskin epithelial cells were isolated, cultured, and grafted as previously described [17]. Tissue fragments were placed in MCDB153-LB culture medium containing 0.25% collagenase, and epithelial outgrowth occurred after 7 to 10 days. Secondary cultures at 40 to 60% confluence were transfected with the recombinant plasmid pMHPV16d or pSHPVlM containing the neomycin resistance gene and entire HPV genomes [17,18]. Immortalized cells were selected by resistance to G418 or by their ability to proliferate with continuous subculturing after normal epithelium had senesced. HPV immortalized cervical and foreskin epithelial cell lines of both early and late passage were used. Early passage refers to cells used prior to 40 population doublings after HPV transfection, and late passage refers to cells cultured for more than 180 population doublings. Nontransfected epithelial cells in secondary or tertiary cultures were utilized for control grafts. Squamous differentiation was induced in culture by allowing cells to become conFIG. 1. Morphologyof xenografts containing normal (A) and HPV fluent and increasing the calcium concentration to 2.0 immortalized cells (B,C). (A) Graft of normal human cervical cells mM to induce intracellular attachment and stratification showing stratified squamous epithelium; (B) graft of HPV-16 immor[17,19]. After further incubation for 3 to 5 days intact talized cervical cells transplanted at later passage; (C) graft of HPVmonolayers were removed from the culture dish by dis- 18 immortalized foreskin cells transplanted at early passage.
KA~IU v r 3rLA3’ “““Z I I\ TRANSFORMATION n ’ nrn -\lnnT
345
traepithelial stages has been suggested by Berkeley [24,25]. While the exact etiologic role of human papillomavirus in the genesis of cervical cancer is still unknown, a strong association of HPV-16 and HPV-18 to cervical cancer has been established. Questions have been raised regarding prognostic significance of HPV types and the possible association of HPV-18 with more biologically aggressive cancers [ 12,161. Other previous clinical studies demonstrate a difference in the observed frequency between HPV-16 and -18 in premalignant and malignant cervical lesions [IS, 161; the observation that RESULTS HPV-18 is much less likely to be seen in a dysplastic A total of 91 epithelial grafts were analyzed. The types lesion as compared to a cancerous lesion is consistent of cells grafted were as follows: normal epithelial cells, with the hypothesis that HPV-18 may be associated with 29; HPV-16 immortalized cells, 36; HPV-18 immortalized lesions that bypass or quickly progress through the incells, 26. Of the grafts utilizing HPV-16 immortalized traepithelial stages. In this study, both HPV-16 and -18 cells, 13 grafts were with early-passage cells and 23 grafts immortalized cell lines produced dysplastic epithelium with late-passage cells. In the HPV-18 immortalized when transplanted in athymic mice. Grafts composed of grafts, 14 utilized early-passage cells and 12 late-passage cultured, immortalized cells of greater than 180 popucells. Microscopic examination of H&E-stained grafts is lation doublings (late passage) formed dysplastic epithedetailed in Table 1 and Fig. 1. Normal epithelial cells lium in approximately equal numbers in HPV-16 and formed stratified squamous tissue in 29 grafts. Of the 36 -18 grafts; however, HPV-18 immortalized cells exhibited grafts utilizing the HPV-16 immortalized cells, 17 formed approximately the same ability to form dysplastic grafts normal-appearing stratified squamous epithelium and 19 utilizing cultured cells of less than 40 population doushowed dysplastic changes. None of the HPV-16 early- blings (early passage) as with late-passage grafts. In conpassage cells resulted in dysplastic changes, while 19 of trast, HPV-16 cell lines produced no dysplastic epithe24 late-passage cell grafts resulted in dysplastic changes. lium in grafts utilizing early-passage cells. This rapid Eighteen of the twenty-six HPV-18 immortalized cell dysplastic transformation of human genital cells by HPVgrafts showed dysplastic changes, with 9 of 14 early- 18 compared to HPV-16 in this in vitro/in vivo system passage grafts and 9 of 11 late-passage grafts showing is consistent with the previous clinical studies suggesting dysplasia. The difference in dysplastic grafts resulting an association between HPV-18 and rapidly progressive from early-passage cells in HPV-16 and -18 grafts is sta- cervical neoplasia. tistically significant (P < 0.0004). No invasion of the underlying basal membrane was observed in either HPVREFERENCES 16 or HPV-18 cell lines. Southern blotting was used to confirm the presence of 1. Rotkin, I. D. A comparison review of key epidemiological studies HPV 16 and 18 in transfected cell lines prior to grafting. in cervical cancer related to current searches for transmissible In addition, in situ hybridization was utilized to confirm agents, Cancer Res. 33 (1973). the presence of HPV messenger RNA in the grafts. Three 2. Kessler, 1. I. Venereal factors in human cervical cancer, Cancer HPV-18 immortalized cell lines which formed dysplastic 39, 1912-1919 (1977). epithelium at early passage expressed moderate to low 3. Lancaster, W. D., and Olson, C. Animal papillomaviruses, Milevels of HPV RNA. Two HPV-16 immortalized cell lines crobio/. Rev. 46, 191-207 (1982). which formed dysplastic epithelium at late passage ex4. Hills, E., and Laverty, C. R. Electron microscopic detection of pressed virus RNA at low to barely detectable levels. papillomavirus particles in selected koilocytotic cells in routine cervical smear, Acta Cytol. 23, 53-58 (1979). Grafts composed of C-41 cells had stronger in situ signals 5. Lancaster, W. D., Kurman, R. J., Sanz, L. E., and Jenson, A. B. than HPV- 16- and HPV- 18-derived grafts. sified as dysplastic: abnormal mitoses, multinucleated cells, increased nuclear-to-cytoplasmic ratio, and absence of basal cell polarity. Graft sections were also mounted on 3-aminopropyltriethoxysilane-coated slides and subjected to in situ to hybridization with wholegenomic tritium-labeled antisense RNA probes for HPV types 16 and 18 [21,22]. In addition, similar grafts using C-41 cervical carcinoma cells were subjected to in situ hybridization [23].
DISCUSSION The clinical presentation and disease course of patients with cervical cancer can vary greatly. As noted by Bain and Cracker, some individuals appear to have a rapid onset of cervical cancer, and a possible distinct subset of cancers with a rapid onset which bypasses the in-
Human papillomavirus: Detection of viral DNA sequences and evidence for molecular heterogeneity in metaplasias and dysplasias of the uterine cervix, Interviro/opy 20, 202-212 (1983). 6. Lancaster, W. D., Castellano, C., Santos, C., Delgado, G., Kurman, R. J., and Jenson, A. B. Human papillomavirus DNA in cervical carcinoma from primary and metastatic sites, Amer. J. Obster. Gynecol. 154, 115-l I8 (1986). 7. Durst, M., Gissmann, L., Ikenberg, H., and zur Hausen, H. A papillomavirus DNA from a cervical carcinoma and its prevalence
346
BARNES
in cancer biopsy samples from different Not/.
Acud.
8. Boshart, M., len, W.. and its presence from cervical
Sci.
USA
80, 3812-3815
geographic
regions, Prac.
(1983).
ET AL. role for type I8 in rapid progression, 159, 293-296 (1988).
Amer.
J. Obstet.
Gynecol.
Gissmann. L., Ikenberg, H., Kleinheinz. A.. Scheurzur Hausen, H. A new type of papillomavirus DNA. in genital cancer biopsies and in cell lines derived cancer, EMEO J. 3, 1151-l 157 (1984).
17. Woodworth, C.. Waggoner, S., Barnes, W., Stoler, M., DePaolo. J. Human cervical and foreskin epithelial cells immortalized by human papillomavirus DNA’s exhibit dysplastic differentiation in Vivo. Cancer Res. 50, 3709-3715 (1990).
9. Lorincz, A. T.. Lancaster, W. D.. and Temple, G. F. Cloning and characterization of the DNA of a new human papillomavirus from a woman with dysplasia of the uterine cervix, J. Viral. 58, 22% 229 (1986).
18. Woodworth, C. D., Bowden, P. E., Doniger. J.. Pirisi, L.. Barnes, 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, 4260-4268 (1988).
IO. de Villiers, E. M.. Gissmann, I... and zur Hausen, H. Molecular cloning of viral DNA from human genital warts, J. Viral. 40, 932935 (1981). Il.
Gissmann. L., Wolnik, L., Ikenberg, H.. Koldovsky. U.. Schnurch. H. G.. and zur Hausen, H. Human papillomavirus types 6 and I I DNA sequences in genital and laryngeal papillomas and in some cervical cancers, Proc. Natl. Acud. Sci. USA 80, 56O563 (1983).
19. Pillai, S., Bilke. D. D., Hincenbergs, M., and Elias, P. M. Biochemical and morphological characterization of growth and differentiation of normal human neonatal keratinocytes in a serum134, 229-237 (1988). free medium, J. Cell. Physiol. 20. Barrandon. Y., Li, D., and Green, H. J. New techniques for the grafting of cultured human epidermal cells onto athymic animals. Inve.st. Dermutol. 91, 315-318 (1988).
12. Reid, R.. Greenberg, M.. Jenson. A. B., et ul. Sexually transmitted papillomaviral infections. I. The anatomic distribution and pathologic grade of neoplastic lesions associated with different viral 156, 212 (1987). types. Amer. J. Oh.stet. Gynrcol.
21. 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. Puthol. 17, 12501259 (1986).
13. Barnes, W., Delgado, G., Kurman, R. J.. et (I/. Possible prognostic significance of human papillomavirus type in cervical cancer, Gynew/. Oncol. 29, 267 (1988).
22. Angerer.
L. M.. Staler. M. H., and Angerer. R. C. in In situ hyApplicutions to neurobiology (K. Valentino, J. Eberwine, and J. Barchus, Eds.). Oxford Univ. Press, New York. pp. 42-70 (1987). hridizution:
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